Extraction of proteins by a two solvent method

ABSTRACT

A method for separating proteins from plant material, in particular, intact algal cells, using an amphipathic solvent set and a hydrophobic solvent set. Some embodiments include dewatering intact algal cells and then extracting proteins from the algal cells. The methods provide for single and multistep extraction processes which allow for efficient separation of algal proteins from a wet algal biomass. These proteins are high value products which can be used as renewable sources of food and food additives. Neutral lipids remaining in the algal biomass after extraction of proteins can be used to generate renewable fuels.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/081,197, filed Apr. 6, 2011, entitled Extraction withFractionation of Oil and Proteinaceous Material from OleaginousMaterial, which claims the benefit of U.S. Provisional Application No.61/321,290, filed Apr. 6, 2010, entitled Extraction with Fractionationof Oil and Proteinaceous Material from Oleaginous Material, and U.S.Provisional Application No. 61/321,286, filed Apr. 6, 2010, entitledExtraction With Fractionation of Oil and Co-Products from OleaginousMaterial, the entire contents of which are hereby incorporated byreference herein.

FIELD OF THE INVENTION

The invention is concerned with extracting and fractionating algalproducts, including, but not limited to, oils and proteins. Inparticular, the methods and systems disclosed herein relate to theseparation of polar lipids. More specifically, the systems and methodsdescribed herein utilize step extraction and fractionation withamphipathic and hydrophobic solvents to process wet algal biomass.

BACKGROUND OF THE INVENTION

Petroleum is a natural resource composed primarily of hydrocarbons.Extracting petroleum oil from the earth is expensive, dangerous, andoften at the expense of the environment. Furthermore, world widereservoirs of oil are dwindling rapidly. Costs also accumulate due tothe transportation and processing required to convert petroleum oil intousable fuels such as gasoline and jet fuel.

Algae have gained a significant importance in recent years given theirability to produce lipids, which can be used to produce sustainablebiofuel. This ability can be exploited to produce renewable fuels,reduce global climate change, and treat wastewater. Algae's superiorityas a biofuel feedstock arises from a variety of factors, including highper-acre productivity compared to typical terrestrial oil crop plants,non-food based feedstock resources, use of otherwise non-productive,non-arable land, utilization of a wide variety of water sources (fresh,brackish, saline, and wastewater), production of both biofuels andvaluable co-products such as carotenoids and chlorophyll.

Several thousand species of algae have been screened and studied forlipid production worldwide over the past several decades. Of these,about 300 species rich in lipid production have been identified. Thelipid composition and content vary at different stages of the life cycleand are affected by environmental and culture conditions. The strategiesand approaches for extraction are rather different depending onindividual algal species/strains employed because of the considerablevariability in biochemical composition and the physical properties ofthe algae cell wall. Conventional physical extraction processes, such asextrusion, do not work well with algae given the thickness of the cellwall and the small size (about 2 to about 20 nm) of algal cells.Furthermore, the large amounts of polar lipids in algal oil, as comparedto the typical oil recovered from seeds, lead to refining issues.

Upon harvesting, typical algal concentrations in cultures range fromabout 0.1-1.0% (w/v). This means that as much as 1000 times the amountof water per unit weight of algae must be removed before attempting oilextraction. Currently, existing oil extraction methods for oleaginousmaterials strictly require almost completely dry feed to improve theyield and quality of the oil extracted. Due to the amount of energyrequired to heat the algal mass to dry it sufficiently, the algal feedto biofuel process is rendered uneconomical. Typically, the feed isextruded or flaked at high temperatures to enhance the extraction. Thesesteps may not work with the existing equipment due to the single cellmicrometric nature of algae. Furthermore, algal oil is very unstable dueto the presence of double bonded long chain fatty acids. The hightemperatures used in conventional extraction methods cause degradationof the oil, thereby increasing the costs of such methods.

It is known in the art to extract oil from dried algal mass by usinghexane as a solvent. This process is energy intensive. The use of heatto dry and hexane to extract produces product of lower quality as thistype of processing causes lipid and protein degradation.

Algal oil extraction can be classified into two types: disruptive ornon-disruptive methods.

Disruptive methods involve lysing cells by mechanical, thermal,enzymatic or chemical methods. Most disruptive methods result inemulsions, requiring an expensive cleanup process. Algal oils contain alarge percentage of polar lipids and proteins which enhance theemulsification of the neutral lipids. The emulsification is furtherstabilized by the nutrient and salt components left in the solution. Theemulsion is a complex mixture, containing neutral lipids, polar lipids,proteins, and other algal products, which extensive refining processesto isolate the neutral lipids, which are the feed that is converted intobiofuel.

Non-disruptive methods provide low yields. Milking is the use ofsolvents or chemicals to extract lipids from a growing algal culture.While sometimes used to extract algal products, milking may not workwith some species of algae due to solvent toxicity and cell walldisruption. This complication makes the development of a generic processdifficult. Furthermore, the volumes of solvents required would beastronomical due to the maximum attainable concentration of the solventin the medium.

Accordingly, to overcome these deficiencies, there is a need in the artfor improved methods and systems for extraction and fractionating algalproducts, in particular algal oil, algal proteins, and algalcarotenoids. This process can be further improved by introducing ahighly non-polar solvent to the extraction system, thereby avoiding thecost of evaporating and recycling all of the solvent used forextraction.

BRIEF SUMMARY OF THE INVENTION

Embodiments described herein relate generally to systems and methods forextracting lipids of varying polarities from an oleaginous material,including for example, an algal biomass. In particular, embodimentsdescribed herein concern extracting lipids of varying polarities from analgal biomass using solvents of varying polarity and/or a series ofmembrane filters. In some embodiments, the filter is a microfilter.

In some embodiments of the invention, a single solvent and water areused to extract and fractionate components present in an oleaginousmaterial. In other embodiments, these components include, but are notlimited to, proteins, polar lipids, and neutral lipids. In still otherembodiments, more than one solvent is used. In still other embodiments,a mixture of solvents is used. The disclosed methods allow forseparation and fractionation of the components of the wet algal biomass.Another aspect of the systems and methods described herein is theability to accomplish preliminary refining, because the hydrophobicsolvent in the system selectively separates the products further intolipids and non-lipid components. The amount of amphipathic solvent usedwill depend on the amount of water in the system. The amount ofhydrophobic solvent is minimized in order to reduce later processing toremove the solvent.

In some embodiments, the methods and systems described herein are usefulfor extracting coproducts of lipids from oleaginous material. Examplesof such coproducts include, without limitation, proteinaceous material,chlorophyll, and carotenoids. Embodiments of the present invention allowfor the simultaneous extraction and fractionation of algal products fromalgal biomass in a manner that allows for the production of both fuelsand nutritional products.

Under one embodiment of the invention, a method for extraction withfractionation of oil and proteinaceous material from oleaginous materialis provided.

Under another embodiment, a method of selectively removing products froman algal biomass comprising substantially intact algal cells includescombining an algal biomass and a first solvent set, to generate a firstextraction mixture, the first extraction mixture including a firstsubstantially solid phase and a first liquid phase; separating at leasta portion of the first liquid phase of the first extraction mixture fromthe first substantially solid phase; combining the first extractionsubstantially solid phase and a second solvent set, to generate a secondextraction mixture, the second extraction mixture including a secondsubstantially solid phase and a second liquid phase, wherein the secondextraction mixture is less polar than the first extraction mixture;separating at least a portion of the second liquid phase of the secondextraction mixture from the second substantially solid phase; combiningthe second extraction substantially solid phase and a third solvent set,to generate a third extraction mixture, the third extraction mixtureincluding a third substantially solid phase and a third liquid phase,wherein the third extraction mixture is less polar than the secondextraction mixture; and separating at least a portion of the thirdliquid phase of the third extraction mixture from the thirdsubstantially solid phase.

In some aspects of the invention, the method further comprises removingat least a portion of the first solvent set from the separated portionof the first liquid phase to obtain a first extraction product. In otheraspects, the method further comprises removing at least a portion of thesecond solvent set from the separated portion of the second liquid phaseto obtain a second extraction product. In still other aspects, themethod further comprises removing at least a portion of the thirdsolvent set from the separated portion of the third liquid phase toobtain a third extraction product.

In yet other aspects, the solvent set comprises a water miscible orwater immiscible solvent. In some aspects, the solvent set comprises twowater miscible or two water immiscible solvents. In other aspects, thesolvent set comprises one or more water miscible solvents and one ormore water immiscible solvents. In still other aspects, the first,second and/or third extraction mixture is heated to a temperature belowits boiling point. In further aspects, the extraction mixture is under apressure greater than atmospheric pressure. In some aspects, at leastone of the first, second, and third solvent sets comprise one or moreamphipathic solvents. In still further aspects, at least one of the oneor more water miscible solvents is selected from the group consisting ofmethanol, ethanol, isopropanol, acetone, ethyl acetate, andacetonitrile. In other aspects, at least one of the first, second, andthird solvent sets comprises ethanol. In still other aspects, thesolvent set is added to the biomass in a 1:1 weight/weight ratio.

In other aspects, the cells comprising the algal biomass are not driedor disrupted. In yet another aspect, the algal biomass is unfrozen. Inanother aspect of the invention, the method further comprises adjustingthe pH of at least one of the first, second, and third extractionmixtures to optimize protein extraction. In still other aspects of theinvention, the algal biomass is simultaneously at least partiallydewatered while products are selectively extracted from the algalbiomass. In yet other aspects of the invention, the first, second, andthird extraction substantially solid phases comprise substantiallyintact algal cells.

Embodiments of the instant invention include a method of separatingpolar lipids from intact algal cells, comprising providing a wet algalbiomass comprising intact algal cells which comprise polar lipids,dewatering the wet algal biomass by removing extracellular water toincrease the solid content of the wet algal biomass to between about 5%w/w and about 50% w/w to result in a partially dewatered wet algalbiomass, mixing the partially dewatered wet algal biomass with anamphipathic solvent set and a hydrophobic solvent set to generate anextraction mixture comprising a heavier phase and a lighter phase,wherein the heavier phase comprises the amphipathic solvent set andpartially dewatered wet algal biomass and the lighter phase comprisesthe hydrophobic solvent set and polar lipids, separating the heavierphase of the extraction mixture from the lighter phase of the extractionmixture, and separating the polar lipids from the lighter phase togenerate a polar lipids fraction.

Some embodiments of the invention include a method of separatingproteins from intact algal cells, comprising providing a wet algalbiomass comprising intact algal cells which comprise water, algalproteins and polar lipids, dewatering the wet algal biomass by removingextracellular water to increase the solid content of the wet algalbiomass to between 5% and 50% to result in a partially dewatered algalbiomass, mixing the partially dewatered wet algal biomass with anamphipathic solvent set and a hydrophobic solvent set to generate anextraction mixture comprising a heavier phase and a lighter phase,wherein the heavier phase comprises the water, amphipathic solvent set,partially dewatered wet algal biomass, and algal proteins, and thelighter phase comprises the hydrophobic solvent set and polar lipids,separating the heavier phase of the extraction mixture from the lighterphase of the extraction mixture, separating the partially dewatered wetalgal biomass from the heavier phase of the extraction mixture togenerate a protein mixture comprising proteins, water and amphipathicsolvent set, and separating the proteins from the protein mixture togenerate a protein fraction.

Still other embodiments include a method of separating proteins fromintact algal cells, comprising providing a wet algal biomass comprisingintact algal cells which comprise water, algal proteins and polarlipids, mixing the wet algal biomass with an amphipathic solvent set anda hydrophobic solvent set to generate an extraction mixture comprising aheavier phase and a lighter phase, wherein the heavier phase comprisesthe water, amphipathic solvent set, wet algal biomass, and algalproteins, and the lighter phase comprises the hydrophobic solvent setand polar lipids, separating the heavier phase of the extraction mixturefrom the lighter phase of the extraction mixture, separating the wetalgal biomass from the heavier phase of the extraction mixture togenerate a protein mixture comprising proteins, water and amphipathicsolvent set, and separating the proteins from the protein mixture togenerate a protein fraction.

Further embodiments of the include a method of separating neutral lipidsfrom intact algal cells, comprising providing a wet algal biomasscomprising intact algal cells which comprise proteins, polar lipids andneutral lipids, mixing the wet algal biomass with a first amphipathicsolvent set and a first hydrophobic solvent set to generate a firstextraction mixture comprising a first heavier phase and a first lighterphase, wherein the first heavier phase comprises the first amphipathicsolvent set, neutral lipids, proteins, and wet algal biomass and thefirst lighter phase comprises the first hydrophobic solvent set and thepolar lipids, separating the first lighter phase of the extractionmixture from the first heavier phase of the extraction mixture,separating the wet algal biomass from the first heavier phase of theextraction mixture to generate a separated wet algal biomass comprisingneutral lipids; and a protein mixture comprising proteins, water and thefirst amphipathic solvent set, mixing the separated algal biomass with asecond amphipathic solvent set and a second hydrophobic solvent set togenerate a second extraction mixture comprising a second heavier phaseand a second lighter phase, wherein the second heavier phase comprisesthe second amphipathic solvent set and separated wet algal biomass andthe second lighter phase comprises the second hydrophobic solvent setand the neutral lipids, separating the second heavier phase from thesecond lighter phase, and separating the neutral lipids from the secondlighter phase to generate a neutral lipids fraction.

Some embodiments further comprise dewatering is carried out bycentrifuging, filtering, settling or float fractionating. Still otherembodiments comprise adding the amphipathic solvent set in an amount toadjust the polarity index of the extraction mixture to between about 6.5to 6.7 prior to the addition of the hydrophobic solvent set. Yet otherembodiments comprise adding the hydrophobic solvent set in an amount toadjust the polarity index of the extraction mixture to about 5.7 to 5.9after the addition of the amphipathic solvent set. In still furtherembodiments, the amphipathic solvent set is acetone, methanol, ethanol,isopropanol, butanone, dimethyl ether, and propionaldehyde. 2-propanol,acetonitrile, t-butyl alcohol, 1-propanol, water, heavy (D₂O), ethyleneglycol, glycerin, or a combination thereof. In yet other embodiments,the hydrophobic solvent set is propane, butane, pentane, butene,propene, naphtha, an alkane, hexane, pentane, heptane, octane, an ester,ethyl acetate, butyl acetate), a ketone, methyl ethyl ketone, methylisobutyl ketone, an aromatic, toluene, benzene, cyclohexane,tetrahydrofuran, a haloalkane, chloroform, trichloroethylene, an ether,diethyl ether, diesel, jet fuel, gasoline, or a combination thereof.

In some embodiments, separating the heavier phase from the lighter phaseincludes decanting, membrane filtering or centrifuging. In otherembodiments, separating the polar lipids from the lighter phase includesevaporation or distillation. In still other embodiments, the hydrophobicsolvent set is recovered. In still other embodiments, the hydrophobicsolvent set is condensed and recovered. On yet others, the amphipathicsolvent set is recovered. In still others, the amphipathic solvent setis condensed and recovered.

In yet other embodiments, the extraction mixture is heated. In furtherembodiments, the extraction mixture is heated with microwaves, water,steam, hot oil or electricity. In some embodiments, the extractionmixture is heated at atmospheric pressure. In still further embodiments,the extraction mixture is heated in a pressurized reactor. In evenfurther embodiments, the pressurized reactor is a batch or a continuousreactor.

Further embodiments include a method of separating neutral lipids fromintact algal cells, comprising providing a wet algal biomass comprisingintact algal cells which comprise proteins, polar lipids and neutrallipids, dewatering the wet algal biomass by removing extracellular waterto increase the solid content of the wet algal biomass to between 5% and50% to generate a partially dewatered wet algal biomass, mixing thepartially dewatered wet algal biomass with a first amphipathic solventset and a first hydrophobic solvent set to generate a first extractionmixture comprising a first heavier phase and a first lighter phase,wherein the first heavier phase comprises the first amphipathic solventset, neutral lipids, proteins, and partially dewatered wet algal biomassand the first lighter phase comprises the first hydrophobic solvent setand the polar lipids, separating the first lighter phase of theextraction mixture from the first heavier phase of the extractionmixture, separating the partially dewatered wet algal biomass from thefirst amphipathic solvent set in the first heavier phase of theextraction mixture to generate a separated partially dewatered wet algalbiomass comprising neutral lipids, and a protein mixture comprisingproteins, water and the first amphipathic solvent set, mixing theseparated partially dewatered algal biomass with a second amphipathicsolvent set and a second hydrophobic solvent set to generate a secondextraction mixture comprising a second heavier phase and a secondlighter phase, wherein the second heavier phase comprises the secondamphipathic solvent set and separated partially dewatered wet algalbiomass and the second lighter phase comprises the second hydrophobicsolvent set and the neutral lipids, separating the second heavier phasefrom the second lighter phase, and separating the neutral lipids fromthe second lighter phase.

In some embodiments, the second lighter phase is heated prior toseparating the neutral lipids from the second lighter phase. In someembodiments, the second hydrophobic solvent set is removed from thesecond lighter phase by evaporation or distillation, thereby generatinga neutral lipid fraction. In other embodiments, at least one of thefirst and second amphipathic solvent set is evaporated. In still otherembodiments, at least one of the first and second hydrophobic solventset is recovered. In yet other embodiments, at least one of the firstand second hydrophobic solvent set is chilled and recovered. In stillfurther embodiments, at least one of the first and second amphipathicsolvent set is chilled and recovered. In further embodiments, at leastone of the first and second amphipathic solvent set is recovered. Instill further embodiments, at least one of the first and secondextraction mixture is heated. In yet other embodiments, at least one ofthe first and second extraction mixture is heated with microwaves,water, steam, or hot oil or electricity. In some embodiments, at leastone of the first and second extraction mixture is heated at atmosphericpressure. In still others, at least one of the first and secondextraction mixture is heated in a pressurized reactor. In someembodiments, the pressurized reactor is a batch or a continuous reactor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a flowchart of steps involved in a method according to anexemplary embodiment of the present disclosure.

FIG. 1B is a schematic diagram of an exemplary embodiment of adewatering process according to the present disclosure.

FIG. 2 is a schematic diagram of an exemplary embodiment of anextraction system according to the present disclosure.

FIG. 3 is a comparative graph showing Sohxlet extraction of freeze driedalgae biomass using an array of solvents encompassing the completepolarity range showing maximum non-disruptive algae oil extractionefficiency and the effect of polarity on the polar and non-polar lipidsextraction.

FIGS. 4A&B are graphic representations showing neutral lipids (A) Purityand (B) Recovery in the two step solvent extraction process usingmethanol and petroleum ether at three different temperatures.

FIGS. 5A&B are graphs showing neutral lipids (A) Purity and (B) Recoveryin the two step solvent extraction process using aqueous methanol andpetroleum ether at three different temperatures.

FIG. 6 is a graph showing lipid recovery in the two step solventextraction process using aqueous methanol and petroleum ether at threedifferent temperatures.

FIG. 7 is a graph showing the effect of solvents to solid biomass ratioon lipid recovery.

FIG. 8 is a graph showing the efficacy of different aqueous extractionsolutions in a single step extraction recovery of aqueous methanol ondry biomass.

FIG. 9 is a graph showing the effect of multiple step methanolextractions on the cumulative total lipid yield and the neutral lipidspurity.

FIG. 10 is a graph showing the cumulative recovery of lipids using wetbiomass and ethanol.

FIG. 11 is a graph showing a comparison of the extraction times of themicrowave assisted extraction and conventional extraction systems.

FIG. 12A is a flowchart of steps involved in a method according to anexemplary embodiment of the present disclosure which incorporates a stepof protein extraction. All of the units in FIG. 12A are in pounds.

FIG. 12B is a flowchart of steps involved in an exemplary extractionprocess according to the present disclosure.

FIG. 13 is a flowchart and mass balance diagram describing one of theembodiments of the present invention wherein 1000 lbs. of algal biomasswas processed through extraction and fractionation in order to separateneutral lipids, polar lipids, and protein from the algal biomass.

FIG. 14 is a flowchart describing one of the embodiments of the presentinvention wherein an algal mass can be processed to form variousproducts.

FIG. 15 is a flowchart describing one of the embodiments of the presentinvention wherein algae neutral lipids are processed to form variousproducts.

FIG. 16 is a flowchart describing one of the embodiments of the presentinvention wherein algae neutral lipids are processed to form fuelproducts.

FIG. 17 is a flowchart describing one of the embodiments of the presentinvention wherein algae proteins are selectively extracted from afreshwater algal biomass.

FIG. 18 is a flowchart describing one of the embodiments of the presentinvention wherein algae proteins are selectively extracted from asaltwater algal biomass.

FIG. 19 is a flowchart describing one of the embodiments of the presentinvention wherein a selected algae protein is extracted from a saltwateror freshwater algal biomass.

FIG. 20 is a flowchart describing one of the embodiments of the presentinvention wherein a selected algae protein is extracted from a saltwateror freshwater algal biomass.

FIG. 21 is a photograph showing Scenedescemus sp. cells before and afterextraction using the methods described herein. The cells aresubstantially intact both before and after extraction.

FIG. 22 is a schematic of an exemplary extraction scheme for theextraction of proteins by a single solvent (amphipathic) system.

FIG. 23 is a schematic of an exemplary extraction scheme for theextraction of polar lipids and proteins by a two-solvent(amphipathic/hydrophobic) system.

FIG. 24 is a schematic of an exemplary extraction scheme for theextraction of neutral lipids and proteins by a two-solvent(amphipathic/hydrophobic) system.

FIG. 25 is a schematic of an exemplary extraction scheme for theextraction of neutral lipids, polar lipids, and proteins by atwo-solvent (amphipathic/hydrophobic) system.

FIG. 26 is a schematic demonstrating the ethanol extraction concept.

DETAILED DESCRIPTION Definitions

The term “conduit” or any variation thereof, as used herein, includesany structure through which a fluid may be conveyed. Non-limitingexamples of conduit include pipes, tubing, channels, or other enclosedstructures.

The term “reservoir” or any variation thereof, as used herein, includesany body structure capable of retaining fluid. Non-limiting examples ofreservoirs include ponds, tanks, lakes, tubs, or other similarstructures.

The term “about” or “approximately,” as used herein, are defined asbeing close to as understood by one of ordinary skill in the art, and inone non-limiting embodiment the terms are defined to be within 10%,preferably within 5%, more preferably within 1%, and most preferablywithin 0.5%.

The terms “inhibiting” or “reducing” or any variation of these terms, asused herein, includes any measurable decrease or complete inhibition toachieve a desired result.

The term “effective,” as used herein, means adequate to accomplish adesired, expected, or intended result.

The use of the word “a” or “an” when used in conjunction with the term“comprising” herein may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

The term “or” as used herein, means “and/or” unless explicitly indicatedto refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.”

The use of the term “wet” as used herein, is used to mean containingabout 50% to about 99.9% water content. Water content may be locatedeither intracellularly or extracelluarly.

The use of the term “solvent set” as used herein, is used to mean acomposition comprising one or more solvents. These solvents can beamphipathic (also known as amphipathic or slightly nonpolar),hydrophilic, or hydrophobic (also known as lipophilic). In someembodiments, these solvents are water miscible and in others, they areimmiscible in water. Non-limiting example of solvents that may be usedto practice the methods of the instant invention include methanol,ethanol, isopropanol, acetone, ethyl acetate, and acetonitrile, alkanes(hexane, pentane, heptane, octane), esters (ethyl acetate, butylacetate), ketones (methyl ethyl ketone (MEK), methyl isobutyl ketone(MIBK)), aromatics (toluene, benzene, cyclohexane, tetrahydrofuran),haloalkanes (chloroform, trichloroethylene), ethers (diethyl ether), andmixtures (diesel, jet fuel, gasoline).

Non-limiting examples of amphipathic solvents include methanol, ethanol,isopropanol, acetone, ethyl acetate, acetonitrile, and combinationsthereof. Food grade ethanols are also desirable for use in the disclosedmethods.

Non-limiting examples of hydrophobic solvents include alkanes (hexane,pentane, heptane, octane), esters (ethyl acetate, butyl acetate),ketones (methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)),aromatics (toluene, benzene, cyclohexane, tetrahydrofuran), haloalkanes(chloroform, trichloroethylene), ethers (diethyl ether), mixtures(diesel, jet fuel, gasoline), and combinations thereof.

Non-limiting examples of solvent recovery methods include evaporation,pervaporation, selective adsorption of the solvent components, steamstripping, and combinations thereof. Pervaporation is the process ofselectively removing the vapors of a solvent component through apolymeric membrane by creating vacuum on the permeate side. Selectiveadsorption is used in the ethanol industry to remove small amounts ofwater from aqueous ethanol using zeolite. Steam stripping can be usedfor non-temperature sensitive operations.

The term “interfacial layer” as used herein means the region comprisingand adjoining the phase boundary within which the polarity index of asolvent set are significantly different from the polarity index ofanother, adjoining solvent set.

The term “polarity index” as used herein refers to the dimensionlessSnyner Polarity Index as described by Snyder, L. R. “Classification ofthe Solvent Properties of Common Liquids” Journal of Chromatography,92(2): 223-230 (May 1974).

The term “oil” as used herein includes compositions containing neutrallipids and polar lipids. The terms “algae oil” and “algal oil” as usedherein are used interchangeably.

The term “diffusate” or “permeate” as used herein may refer to materialthat has passed through a separation device, including, but not limitedto a filter or membrane.

The term “retentate” as used herein may refer to material that remainsafter the diffusate has passed through a separation device.

As used herein, the words “comprising” (and any form of comprising, suchas “comprise” and “comprises”), “having” (and any form of having, suchas “have” and “has”), “including” (and any form of including, such as“includes” and “include”), or “containing” (and any form of containing,such as “contains” and “contain”) are inclusive or open-ended and do notexclude additional, unrecited elements or method steps.

The term “polar lipids” or any variation thereof, as used herein,includes, but is not limited to, phospholipids and glycolipids.

The term “neutral lipids” or any variation thereof, as used herein,includes, but is not limited to, triglycerides, diglycerides,monoglycerides, carotenoids, waxes, sterols.

The term “solid phase” as used herein refers to a collection of materialthat is generally more solid than not, and is not intended to mean thatall of the material in the phase is solid. Thus, a phase having asubstantial amount of solids, while retaining some liquids, isencompassed within the meaning of that term. Meanwhile, the term “liquidphase”, as used herein, refers to a collection of material that isgenerally more liquid than not, and such collection may include solidmaterials.

The term “biodiesel” as used herein refers to methyl or ethyl esters offatty acids derived from algae

The term “nutraceutical” as used herein refers to a food product thatprovides health and/or medical benefits. Non-limiting examples includecarotenoids, carotenes, xanthophylls such as zeaxanthin, astaxanthin,and lutein.

The term “biofuel” as used herein refers to fuel derived from biologicalsource. Non-limiting examples include biodiesel, jet fuel, diesel, jetfuel blend stock and diesel blend stock.

The term “impurities”, when used in connection with polar lipids, asused herein, refers to all components other than the products ofinterest that are coextracted or have the same properties as the productof interest.

The term “lubricants”, when used in connection with polar lipids, asused herein refers to hydrotreated algal lipids such as C16-C20 alkanes.

The term “detergents”, when used in connection with polar lipids, asused herein refers to glycolipids, phospholipids and derivativesthereof.

The term “food additives”, when used in connection with polar lipids, asused herein refers to soy lecithin substitutes or phospholipids derivedfrom algae.

The term “non-glycerin matter” as used herein refers to any impuritythat separates with the glycerin fraction. A further clean up step willremove most of what is present in order to produce pharmaceutical gradeglycerin.

The term “unsaturated fatty acids” as used herein refers to fatty acidswith at least one double carbon bond. Non-limiting examples ofunsaturated fatty acids include palmitoleic acid, margaric acid, stearicacid, oleic acid, octadecenoic acid, linoleic acid, gamma-linoleic acid,alpha linoleic acid, arachidic acid, eicosenoic acid, homogamma linoleicacid, arachidonic acid, eicosapenenoic acid, behenic, docosadienoicacid, heneicosapentaenoic, docosatetraenoic acid. Fatty acids having 20or more carbon atoms in the backbone are generally referred to as “longchain fatty acids”. The fatty acids having 19 or fewer carbon atoms inthe backbone are generally referred to as “short chain fatty acids”.

Unsaturated long chain fatty acids include, but are not limited to,omega-3 fatty acids, omega-6 fatty acids, and omega-9 fatty acids. Theterm “omega-3 fatty acids” as used herein refers to, but is not limitedto the fatty acids listed in Table 1.

TABLE 1 Lipid Common name name Chemical name Eicosatrienoic acid (ETE)20:3 (n−3) all-cis-11,14,17- eicosatrienoic acid Eicosatetraenoic acid(ETA) 20:4 (n−3) all-cis-8,11,14,17- eicosatetraenoic acidEicosapentaenoic acid (EPA) 20:5 (n−3) all-cis-5,8,11,14,17-eicosapentaenoic acid Heneicosapentaenoic acid (HPA) 21:5 (n−3)all-cis-6,9,12,15,18- heneicosapentaenoic acid Docosapentaenoic acid(DPA), 22:5 (n−3) all-cis-7,10,13,16,19- docosapentaenoic acidClupanodonic acid 22:6 (n−3) all-cis-4,7,10,13,16,19- docosahexaenoicacid Docosahexaenoic acid (DHA) 24:5 (n−3) all-cis-9,12,15,18,21-tetracosapentaenoic acid Tetracosapentaenoic acid 24:6 (n−3)all-cis-6,9,12,15,18,21- tetracosahexaenoic acid

The term “jet fuel blend stock” as used herein refers to alkanes withthe carbon chain lengths appropriate for use as jet fuels.

The term “diesel blend stock” as used herein refers to alkanes with thecarbon chain lengths appropriate for use as diesel.

The term “animal feed” as used herein refers to algae-derived substancesthat can be consumed and used to provide nutritional support for ananimal.

The term “human food” as used herein refers to algae-derived substancesthat can be consumed to provide nutritional support for people.Algae-derived human food products can contain essential nutrients, suchas carbohydrates, fats, proteins, vitamins, or minerals.

The term “bioremediation” as used herein refers to use of algal growthto remove pollutants, such as, but not limited to, nitrates, phosphates,and heavy metals, from industrial wastewater or municipal wastewater.

The term “wastewater” as used herein refers to industrial wastewater ormunicipal wastewater that contain a variety of contaminants orpollutants, including, but not limited to nitrates, phosphates, andheavy metals.

The term “enriched”, as used herein, shall mean about 50% or greatercontent.

The term “substantially”, as used herein, shall mean mostly.

The term “globulin proteins” as used herein refers to salt solubleproteins.

The term “albumin proteins” as used herein refers to water solubleproteins.

The term “glutelin proteins” as used herein refers to alkali solubleproteins.

The term “prolamin proteins” as used herein refers to alcohol solubleproteins. Non-limiting examples of prolamin proteins are gliadin, zein,hordein, avenin.

The term “algal culture” as used herein refers to algal cells in culturemedium.

The term “algal biomass” as used herein refers to an at least partiallydewatered algal culture.

The term “dewatered” as used herein refers to the removal of at leastsome water.

The term “algal paste” as used herein refers to a partially dewateredalgal culture having fluid properties that allow it to flow. Generallyan algal paste has a water content of about 90%.

The term “algal cake” as used herein refers to a partially dewateredalgal culture that lacks the fluid properties of an algal paste andtends to clump. Generally an algal cake has a water content of about 60%or less.

Saltwater algal cells include, but are not limited to, marine andbrackish algal species. Saltwater algal cells are found in nature inbodies of water such as, but not limited to, seas, oceans, andestuaries. Non-limiting examples of saltwater algal species includeNannochloropsis sp., Dunaliella sp.

Freshwater algal cells are found in nature in bodies of water such as,but not limited to, lakes and ponds. Non-limiting examples of freshwateralgal species include Scendescemus sp., Haemotococcus sp.

Non-limiting examples of microalgae that can be used with the methods ofthe invention are members of one of the following divisions:Chlorophyta, Cyanophyta (Cyanobacteria), and Heterokontophyta. Incertain embodiments, the microalgae used with the methods of theinvention are members of one of the following classes:Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In certainembodiments, the microalgae used with the methods of the invention aremembers of one of the following genera: Nannochloropsis, Chlorella,Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium,Spirulina, Amphora, and Ochromonas.

Non-limiting examples of microalgae species that can be used with themethods of the present invention include: Achnanthes orientalis,Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphoracoffeiformis var. linea, Amphora coffeiformis var. punctata, Amphoracoffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphoradelicatissima, Amphora delicatissima var. capitata, Amphora sp.,Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekeloviahooglandii, Borodinella sp., Botryococcus braunii, Botryococcussudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria,Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var.subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorellaanitrata, Chlorella antarctica, Chlorella aureoviridis, ChlorellaCandida, Chlorella capsulate, Chlorella desiccate, Chlorellaellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var.vacuolate, Chlorella glucotropha, Chlorella infusionum, Chlorellainfusionum var. actophila, Chlorella infusionum var. auxenophila,Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis,Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var.lutescens, Chlorella miniata, Chlorella minutissima, Chlorellamutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva,Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides,Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorellaregularis var. minima, Chlorella regularis var. umbricata, Chlorellareisiglii, Chlorella saccharophila, Chlorella saccharophila var.ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana,Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorellavanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorellavulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorellavulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia,Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella,Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris,Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonassp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.,Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliellagranulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva,Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliellaterricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliellatertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp.,Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp.,Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonassp., Isochrysis aff. galbana, lsochrysis galbana, Lepocinclis,Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp.,Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Naviculaacceptata, Navicula biskanterae, Navicula pseudotenelloides, Naviculapelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp.,Nephroselmis sp., Nitschia communis, Nitzschia alexandrine, Nitzschiaclosterium, Nitzschia communis, Nitzschia dissipata, Nitzschiafrustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschiaintermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusillaelliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular,Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla,Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoriasubbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp.,Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp.,Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis,Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica,Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte,Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis,Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta,Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica,Thalassiosira weissflogii, and Viridiella fridericiana.

In other embodiments, the biomass can be plant material, including butnot limited to soy, corn, palm, camelina, jatropha, canola, coconut,peanut, safflower, cottonseed, linseed, sunflower, rice bran, and olive.

Systems and methods for extracting lipids and coproducts (e.g.,proteins) of varying polarity from a wet oleaginous material, includingfor example, an algal biomass, are disclosed. In particular, the methodsand systems described herein concern the ability to both extract andfractionate the algae components by doing sequential extractions with anamphipathic/hydrophobic solvent and water mixture that becomesprogressively less polar (i.e., water in solvent/water ratio isprogressively reduced as one proceeds from one extraction step to thenext). In other words, the interstitial water initially present in thealgal in the algae (approximately 75% of its weight) is graduallyreplaced by the amphipathic/hydrophobic solvent to the azeotrope of theamphipathic/hydrophobic solvent. This results in the extraction ofcomponents soluble at the polarity developed at each step, therebyleading to simultaneous fractionation of the extracted components.

This process leads to a mixture containing two phases, one lighter andone heavier. The lighter phases comprises the hydrophobic solvent andthe polar lipids. Nonpolar lipids are also present in this phase. Theheavier phase comprises the amphipathic solvent, water, and theremaining algal biomass. The presence of the hydrophobic solvent allowsfor selective extraction of the lipid components from the other algalcomponents present in the total extract. This results in purer lipidproduct with almost no protein or water soluble algal product content.

In some embodiments of the invention, a single solvent and water areused to extract and fractionate components present in an oleaginousmaterial. In other embodiments, a solvent set and water are used toextract and fractionate components present in an oleaginous material. Insome embodiments the oleaginous material is wet. In other embodiments,the oleaginous material is algae.

Polar lipid recovery depends mainly on its ionic charge, watersolubility, and location (intracellular, extracellular or membranebound). Examples of polar lipids include, but are not limited to,phospholipids and glycolipids. Strategies that can be used to separateand purify polar lipids can roughly be divided into batch or continuousmodes. Examples of batch modes include precipitation (pH, organicsolvent), solvent extraction and crystallization. Examples of continuousmodes include centrifuging, adsorption, foam separation andprecipitation, and membrane technologies (tangential flow filtration,diafiltration and precipitation, ultra filtration).

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the examples,while indicating specific embodiments of the invention, are given by wayof illustration only. Additionally, it is contemplated that changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

Surprisingly, the proposed non-disruptive extraction process results inover 90% recovery. The small amount of polar lipids in the remainingbiomass enhances its value when the remaining biomass is used for feed.This is due, at least in part, to the high long chain unsaturated fattyacid content of the biomass. In addition, ethanol extracts can furtherbe directly transesterified. Furthermore, unlike the existingconventional methods, the methods and systems described herein aregeneric for any algae, and enable recovery of a significant portion ofthe valuable components, including polar lipids, in the algae by the useof a water miscible organic solvent gradient.

The neutral lipid fraction obtained by the use of the present inventionpossesses a low metal content, thereby enhancing stability of the lipidfraction, and reducing subsequent processing steps. Metals tend to makeneutral lipids unstable due to their ability to catalyze oxidation.Furthermore, metals inhibit hydrotreating catalysts, necessitating theirremoval before a neutral lipid mixture can be refined. The systems andmethods disclosed herein allow for the extraction of metals in theprotein and/or the polar lipid fractions. This is advantageous becauseproteins and polar lipids are not highly affected by metal exposure, andin some cases are actually stabilized by metals.

The systems and methods disclosed herein can start with wet biomass,reducing the drying and dewatering costs. Compared to conventionalextraction processes, the disclosed extraction and fractionationprocesses should have relatively low operating costs due to the moderatetemperature and pressure conditions, along with the solvent recycle.Furthermore, conventional extraction processes are cost prohibitive andcannot meet the demand of the market.

Another aspect of the systems and methods described herein is theability to accomplish preliminary refining, which is the separation ofpolar lipids from neutral lipids during the extraction process. Thedifferences between algal oil used in exemplary embodiments andvegetable oils used in previous embodiments include the percentage ofindividual classes of lipids. An exemplary algal crude oil compositionis compared with vegetable oil shown in Table 2 below:

TABLE 2 Algal Crude Oil (w/w) Vegetable Oil (w/w) Neutral lipids 30-90%90-98%   Phospholipids 10-40% 1-2%  Glycolipids 10-40% <1% Free fattyacids  1-10% <3% Waxes  2-5% <2% Pigments  1-4% Ppm

Degumming (physical and/or chemical) of vegetable oil is done in orderto remove polar lipids (e.g., glycolipids and phospholipids). Vegetableoil that has been chemically degummed retains a significant quantity ofneutral lipid. This neutral lipid fraction is further removed from thedegummed material using solvent extraction or supercritical/subcriticalfluid extraction or membrane technology. In contrast, separation of theneutral lipids from an oleaginous algal biomass is far more difficultthan from a vegetable oil feedstock due to the presence of largequantities of polar lipids typically found in algal oil (see Table 2).This is because the larger percentage of polar lipids present in algaloil enhances the emulsification of the neutral lipids. Theemulsification is further stabilized by the nutrient and salt componentsleft in the solution. The presence of polar lipids, along with metals,results in processing difficulties for separation and utilization ofneutral lipids. However, because polar lipids have an existing market,their recovery would add significant value to the use of algal oil togenerate fuels.

Polar lipids are surfactants by nature due to their molecular structureand have a huge existing market. Many of the existing technologies forproducing polar lipids are raw material or cost prohibitive. Alternativefeedstocks for glycolipids and phospholipids are mainly algae oil, oatoil, wheat germ oil and vegetable oil. Algae oil typically containsabout 30-85% (w/w) polar lipids depending on the species, physiologicalstatus of the cell, culture conditions, time of harvest, and the solventutilized for extraction. Further, the glycerol backbone of each polarlipid has two fatty acid groups attached instead of three in the neutrallipid triacylglycerol. Transesterification of polar lipids may yieldonly two-thirds of the end product, i.e., esterified fatty acids, ascompared to that of neutral lipids, on a per mass basis. Hence, removaland recovery of the polar lipids would not only be highly beneficial inproducing high quality biofuels or triglycerides from algae, but alsogenerate value-added co-products glycolipids and phospholipids, which inturn can offset the cost associated with algae-based biofuel production.The ability to easily recover and fractionate the various oil andco-products produced by algae is advantageous to the economic success ofthe algae oil process.

A further aspect of the methods and systems described herein is theability to extract proteins from an oleaginous material, such as algalbiomass. The methods disclosed herein of extraction of proteinaceousmaterial from algal biomass comprise a flexible and highly customizableprocess of extraction and fractionation. For example, in someembodiments, extraction and fractionation occur in a single step,thereby providing a highly efficient process. Proteins sourced from suchbiomass are useful for animal feeds, food ingredients and industrialproducts. For example, such proteins are useful in applications such asfibers, adhesives, coatings, ceramics, inks, cosmetics, textiles,chewing gum, and biodegradable plastics.

Another aspect of the methods and systems described herein involvesvarying the ratio of algal biomass to solvent based on the components tobe extracted. In one embodiment, an algal biomass is mixed with an equalweight of solvent. In another embodiment, an algal biomass is mixed witha lesser weight of solvent. In yet another embodiment, an algal biomassis mixed with a greater weight of solvent. In some embodiments, theamount of solvent mixed with an algal biomass is calculated based on thesolvent to be used and the desired polarity of the algal biomass/solventmixture. In still other embodiments, the algal mass is extracted inseveral steps. In an exemplary embodiment, an algal biomass issequentially extracted, first with about 50-60% of its weight with aslightly nonpolar, water miscible solvent. Second, the remaining algalsolids are extracted using about 70% of the solids' weight in solvent. Athird extraction is then performed using about 90% of the solid's weightin solvent. Having been informed of these aspects of the invention, oneof skill in the art would be able to use different solvents of differentpolarities by adjusting the ratios of algal biomass and/or solidresiduals to the desired polarity in order to selectively extract algalproducts.

For example, in preferred embodiment, the solvent used is ethanol.Components may be selectively isolated by varying the ratio of solvent.Proteins can be extracted from an algal biomass with about 50% ethanol,polar lipids with about 80% ethanol, and neutral lipids with about 95%or greater ethanol. If methanol were to be used, the solventconcentration to extract proteins from an algal biomass would be about70%. Polar lipids would require about 90% methanol, and neutral lipidswould require about 100% methanol.

Embodiments of the systems and methods described herein exhibitsurprising and unexpected results. First of all, the recovery/extractionprocess can be done on a wet biomass. This is a major economic advantageas exemplary embodiments avoid the use of large amounts of energyrequired to dry and disrupt the cells. Extraction of neutral lipids froma dry algal biomass is far more effective using the systems and methodsof the present invention. The yields obtained from the disclosedprocesses are significantly higher and purer than those obtained byconventional extractions. This is because conventional extractionfrequently results in emulsions, rendering component separationsextremely difficult.

Exemplary embodiments may be applied to any algae or non-algaeoleaginous material. Exemplary embodiments may use any water-miscibleslightly nonpolar solvent, including, but not limited to, methanol,ethanol, isopropanol, acetone, ethyl acetate, and acetonitrile. Specificembodiments may use a green renewable solvent, such as ethanol. Thealcohol solvents tested resulted in higher yield and purity of isolatedneutral lipids. Ethanol is relatively economical to purchase as comparedto other solvents disclosed herein. In some exemplary embodiments,extraction and fractionation can be performed in one step followed bymembrane-based purification if needed. The resulting biomass is almostdevoid of water and can be completely dried with lesser energy than anaqueous algae slurry.

In some exemplary embodiments, the solvent used to extract is ethanol.Other embodiments include, but are not limited to, cyclohexane,petroleum ether, pentane, hexane, heptane, diethyl ether, toluene, ethylacetate, chloroform, dicholoromethane, acetone, acetonitrile,isopropanol, and methanol. In some embodiments, the same solvent is usedin sequential extraction steps. In other embodiments, different solventsare used in each extraction step. In still other embodiments, two ormore solvents are mixed and used in one or more extraction steps.

In some embodiments of the methods described herein, a mixture of two ormore solvents used in any of the extraction steps includes at least onehydrophilic solvent and at least one hydrophobic solvent. When usingsuch a mixture, the hydrophilic solvent extracts the material from thebiomass via diffusion. Meanwhile, a relatively small amount ofhydrophobic solvent is used in combination and is involved in aliquid-liquid separation such that the material of interest isconcentrated in the small amount of hydrophobic solvent. The twodifferent solvents then form a two-layer system, which can be separatedusing techniques known in the art. In such an implementation, thehydrophobic solvent can be any one or more of an alkane, an ester, aketone, an aromatic, a haloalkane, an ether, or a commercial mixture(e.g., diesel, jet fuel, gasoline).

In some embodiments, the extraction processes described hereinincorporate pH excursion in one or more steps. Such pH excursion isuseful for isolating proteinaceous material. In some embodiments, the pHof the extraction process is acid (e.g., less than about 5). In someembodiments, the pH of the extraction process is alkaline (e.g., greaterthan about 10).

The use of hexane in conventional extraction procedures contaminatesalgal biomass such that coproducts may not be used in food products.Embodiments of the present invention are superior to those known in theart as they require the use of far less energy and render productssuitable for use as fuels as well as foodstuffs and nutrientsupplements.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or system of theinvention, and vice versa. Furthermore, systems of the invention can beused to achieve methods of the invention.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

For solvent extraction of oil from algae the best case scenario is asolvent which selectively extracts triacylglycerols (TAG) and leavingall polar lipids and non-TAG neutral lipids such as waxes, sterols inthe algal cell with high recoveries. The second option would beselectively extract polar lipids and then extract purer neutral lipidsdevoid of polar lipids, resulting in high recovery. The last optionwould be to extract all the lipids and achieve very high recovery in oneor two steps.

Referring now to FIG. 1A, a flowchart 100 provides an overview of thesteps involved in exemplary embodiments of methods used in thefractionation and purification of lipids from an algae-containingbiomass. In a first step 110, algal cells are harvested. In a subsequentstep 120, water is removed from algal cells to yield a 10-25% solidbiomass. In step 130, a solvent-based extraction is performed on thebiomass and the fractions are collected. In some embodiments, step 130will also incorporate pH-based extraction and fraction collection.Finally, a solid/liquid phase separation, including, but not limited totechniques such as filtration, decanting, and centrifugation, may beperformed in a step 140 to in order to separate out smaller lipidcomponents.

The algae biomass when harvested in step 110 typically consists of about1-5 g/L of total solids. The biomass can be partially dewatered in step120 using techniques including, but not limited to, dissolved airfloatation, membrane filtration, flocculation, sedimentation, filterpressing, decantation or centrifugation. Dewatering is the removal ofsome, most, or all of the water from a solid or semisolid substance.Embodiments of the present invention utilize dewatering techniques toremove water from a harvested algal biomass. Dewatering can be carriedout using any one of or a combination of any of the methods describedherein, as well as by any other methods known to one of skill in theart.

The dewatered algae biomass resulting from step 120 typically consistsof about 10-30% solids. This biomass can then be extracted with watermiscible slightly nonpolar solvents (e.g., alcohols), in a multistagecountercurrent solvent extraction process segregating the fractions ateach stage. This type of process can reduce both capital and operatingexpenses. In some embodiments, the biomass also undergoes acid and/oralkaline extraction to fractionate protein material.

In some embodiments, dewatering of an algal biomass can be carried outby treating the harvested algal biomass with a solvent such as ethanol.The algal biomass is then allowed to settle out of solution and theliquids may then be removed by methods such as, but not limited to,siphoning. This novel method of dewatering has lower capital andoperating costs than known methods, enables solvent recycling, reducesthe cost of drying the biomass, and has the added benefit of decreasingthe polarity of the algal biomass prior to beginning extraction and/orseparation of algal components. In fact, it is theorized that thesolvent-based sedimentation processes described herein are effective, inpart, due to the fact that organic solvents reduce or neutralize thenegative charge on the algae surface. In some embodiments of theinvention, dewatering methods are combined in order to remove even morewater. In some embodiments, the addition of solvent during thedewatering process begins the process of extraction.

FIG. 1B shows an illustrative implementation of a dewatering process300. An algal culture 310 having a final dry weight of about 1 g/L toabout 10 g/L (i.e., 0.1-1% w/w) is subjected to a water separationprocess 320. Process 320 can include centrifugation, decanting,settling, or filtration. In one embodiment, a sintered metal tube filteris used to separate the algal biomass from the water of the culture.When using such a filter, the recovered water 330 is recycled directedto other algae cultures. Meanwhile, the algal biomass recovered has beenconcentrated to an “algae paste” with a algae density as high as about200 g/L (i.e., 10-20% w/w). This concentrated algae paste is thentreated with a solvent 340 in a solvent-based sedimentation process 350.

Sedimentation process 350 involves adding solvent 340 to the algae pasteto achieve a mixture having a weight/weight solvent to biomass ratio ofbetween about 1:1 to about 1:10. The algae is allowed to settle in asettling vessel, and a solvent/water mixture 360 is removed by, forexample, siphoning and/or decanting. The solvent can be recovered andreused by well-known techniques, such as distillation and/orpervaporation. The remaining wet biomass 370 is expected to have asolids content of about 30% to about 60% w/w in an alcohol and watersolution.

Solvents ideal for dewatering are industrially common water-solublesolvents with densities over 1.1 g/mL or below 0.9 g/mL. Examplesinclude isopropanol, acetone, acetonitrile, t-butyl alcohol, ethanol,methanol, 1-propanol, heavy water (D₂O), ethylene glycol, and/orglycerin. If the solvent density is over 1.1 g/mL then the algae biomasswould float rather than create a sediment at the bottom of the settlingvessel.

FIG. 2 is a schematic diagram of an exemplary embodiment of anextraction system 200. The wet or dry algal biomass is transported usingmethods known in the art, including, but not limited to a moving belt, ascrew conveyor, or through extraction chambers. The solvent forextraction is recirculated from a storage tank assigned to each biomassslot position. The extraction mixture is filtered, returning the biomasssolids back into the slot and the extract into the storage tank. Thesolids on the belt move periodically based on the residence timerequirement for extraction. The extracts in each storage tank may eitherbe replenished at saturation or continuously replaced by fresh solvent.This would also reduce the downstream processing time and costdrastically. This embodiment comprises a primary reservoir 210, atransport mechanism 220, a plurality of separation devices 241-248(e.g., membrane filtration devices), a plurality of extractionreservoirs 261-268, and a plurality of recycle pumps 281-287. In thisembodiment, primary reservoir 210 is divided up into a plurality ofinlet reservoirs 211-218.

During operation, algal biomass 201 is placed a first inlet reservoir211 near a first end 221 of transport mechanism 220. In addition,solvent 205 is placed into inlet reservoir 218 near a second end 222 oftransport mechanism 220. Transport mechanism 220 directs the algalbiomass along transport mechanism 220 from first end 221 towards secondend 222. As the algal biomass is transported, it passes through theplurality of separation devices 241-248 and is separated into fractionsof varying polarity. The diffusate portions that pass through separationdevices 241-248 are directed to reservoirs 261-268.

For example, the diffusate portion of the algal biomass that passesthrough the first separation device 241 (e.g., the portion containingliquid and particles small enough to pass through separation device 241)is directed to the first reservoir 261. From first reservoir 261, thediffusate portion can be recycled back to first inlet reservoir 201. Theretentate portion of the algal biomass that does not pass through firstseparation device 241 can then be directed by transport mechanism 220 tosecond inlet reservoir 212 and second separation device 242, which cancomprise a finer separation or filtration media than the firstseparation device 241.

The segment of the diffusate portion that passes through secondseparation device 242 can be directed to second reservoir 262, and thenrecycled back to second inlet reservoir 212 via recycle pump 282. Theretentate or extracted portion of the algal biomass that does not passthrough second separation device 242 can be directed by transportmechanism 220 to third inlet reservoir 213. This process can be repeatedfor inlet reservoirs 213-218 and separation devices 243-248 such thatthe retentate portions at each stage are directed to the subsequentinlet reservoirs, while the diffusate portions are directed to therecycle reservoirs and recycled back to the current inlet reservoir.

In exemplary embodiments, the first fraction will be extracted with thehighest water to slightly nonpolar solvent ratio, i.e., most polarmixture, while the last fraction will be extracted with the most pureslightly nonpolar solvent, i.e. the least polar mixture. The processtherefore extracts components in the order of decreasing polarity withthe fraction. The function of the first fraction is to remove theresidual water and facilitate the solvent extraction process. Thefractions that follow are rich in polar lipids, while the finalfractions are rich in neutral lipids.

The oil fraction can be esterified to liberate the long chainunsaturated fatty acids. The carotenoids and long chain unsaturatedfatty acids can be separated from the oil using processes such asmolecular distillation in conjunction with non-molecular distillation.All of the fatty acids can be separated from the carotenoids using themolecular distillation. The distillates can be fractionated using asimple distillation column to separate the lower chain fatty acids forrefining. The long chain unsaturated fatty acids remain as high boilingresidue in the column.

In some non-limiting embodiments, the extraction system and methodsdescribed herein incorporate one or more steps to isolate proteinmaterial from the oleaginous material (e.g., algal biomass). Suchprotein extraction steps employ pH adjustment(s) to achieve isolationand extraction of protein. For example, in one non-limiting embodiment,the pH of the solvent in the first separation device is optimized forprotein extraction, resulting in a first fraction that is rich inprotein material. The pH of the protein extraction step is adjusteddepending on the pKa of the proteins of interest. The pKa of a proteinof interest may be ascertained using methods known to one of skill inthe art, including, but not limited to using the Poisson-Boltzmannequation, empirical methods, molecular dynamics based methods, or theuse of titration curves.

In some embodiments, the solvent pH is alkaline. For example, in someembodiments, the solvent pH is greater than about 10. In otherembodiments, the solvent pH ranges from about 10 to about 12. In furtherembodiments, the solvent pH is about 10, about 11, or about 12. In otherembodiments, the solvent pH is acid. For example, in some embodiments,the solvent pH is less than about 5. In other embodiments, the solventpH ranges from about 2 to about 5. In further embodiments, the solventpH is about 2, about 3, about 4, about 4.5, or about 5. The extractedportion of the first separation device is then directed to subsequentinlet reservoirs to achieve extraction and fractionation based onpolarity. In another non-limiting embodiment, protein material isseparated in the final separation device by similar means (i.e., solventpH adjustment).

Adjustment of solvent pH is accomplished in accordance with methodsknown to those of skill in the art. For example, acid pH is achieved bymixture of an appropriate acid into the solvent stream. Exemplary acidsuseful for protein extraction include, without limitation, phosphoricacid, sulfuric acid, and hydrochloric acid. Similarly, alkaline pH isachieved by addition and mixture of an appropriate base into the solventstream. Exemplary bases useful for protein extraction include, withoutlimitation, potassium hydroxide, and sodium hydroxide.

In some embodiments, protein extraction is performed in a systemseparate from the extraction and fractionation system described herein.For example, in some embodiments, an algal biomass is soaked in apH-adjusted solvent mixture, followed by isolation via an appropriateseparation technique (e.g., centrifugation, or filtration). Theremaining solid is then introduced into an extraction and fractionationsystem based on polarity, as described herein. Similarly, in someembodiments, the remaining extract from an extraction and fractionationprocess based on polarity is exposed to a pH-adjusted solvent mixture toisolate protein material at the end of the extraction process.

As shown in FIG. 3, the solvent selection and the theory offractionation based on polarity were developed by extensive analysis ofsolvents and the effect on extraction using the Sohxlet extractionprocess, which allows the separation of lipids from a solid material.The Sohxlet extraction system was utilized for rapid screening solventsfor lipid class selectivity and recovery. Solvents from various chemicalclasses encompassing a wide range of polarities such as alkanes,cycloalkane, alkyl halides, esters, ketones, were tested. Prior to theextraction, the lipid content and composition of the biomass to beextracted was tested in triplicate using the standard methods for algaeoil estimation such as the Bligh-Dyer lipid extraction method. Thebiomass contained 22.16% total lipid, of which 49.52% was neutral lipid.

FIG. 3 presents the data gathered by extraction of a dry algal massusing various polar and nonpolar solvents combined with a Sohxletextraction process. Depending on the chain length of the alkane solvent,60-70% purity of neutral lipids and 15-45% of total lipid recovery canbe achieved without disruption and solvent extraction. The longest chainalkane solvent tested, heptane, recovered 60% of the neutral lipids and42% of the total lipid. As FIG. 3 shows, the results of extraction ofdry algal mass using solvents and conventional extraction methods suchas hexane are inefficient, expensive, and result in poor yields. Thesystems and methods discloses herein address these inefficiencies bycontrolling the proportion of slightly nonpolar solvent to water inorder to separate out components of differing polarities with minimalloss of components.

The lower carbon alcohol solvents were more selective for polar lipids.The neutral lipid purity was 22% for methanol and 45% for ethanol.Isopropyl alcohol did not show any selectivity between polar andnonpolar lipids, resulting in a 52% pure neutral lipid product. Methanolrecovered 67% of the total lipids and more than 90% of the polar lipids.Therefore, methanol is an excellent candidate for an embodiment of thepresent invention wherein methanol can be used to selectively extractpolar lipids from an oleaginous material prior to extracting the neutrallipids using heptane or hexane. The other solvent classes tested did notshow any selectivity towards lipid class since the neutral lipid puritywas close to 49%, similar to the lipid composition present in theoriginal biomass. Furthermore, the total lipid recovery achieved withthese solvents ranged from about 15-35%, rendering these solventsunsuitable for the selective extraction of particular lipid classes ortotal lipid extraction.

The results from the Sohxlet analysis were confirmed using the standardbench scale batch solvent extraction apparatus described below inExample 1. The solvents selected were methanol for the first step torecover polar lipids, and petroleum ether in the second step forrecovery of neutral lipids. All of the extractions were performed with a1:10 solid:solvent ratio. Each extraction step in this experiment was 1hour long. Other experiments done (data not shown) indicate that about45 minutes or longer is long enough for the extraction to be successful.This retention time is dependent on the heat and mass transfer of thesystem.

The methanol extractions were performed at different temperatures, 40°C., 50° C., and 65° C., in order to determine which was optimal. Thepetroleum ether extraction was performed at 35° C., close to the boilingpoint of the solvent. Petroleum ether was chosen because of its highselectivity for neutral lipids, low boiling point, and the productquality observed after extraction.

FIG. 4A shows that the neutral lipid purity in a petroleum etherextraction carried out after a methanol extraction step at 65° C. isover 80%, demonstrating that the combination of these two extractionsteps enhanced the neutral lipid content of the final crude oil product.FIG. 4B shows that the total neutral lipid recovery was low and therewas a significant amount of neutral lipid loss in the first step.

To minimize the loss of neutral lipids in the methanol extraction step,the polarity of the solvent can be increased by adding water to thesolvent. FIGS. 5A and 5B show the results of extracting theaforementioned biomass with 70% v/v aqueous methanol followed byextraction with petroleum ether. FIG. 5A shows that the neutral lipidpurity was much higher in the petroleum ether extraction than wasachieved by the use of pure methanol. Moreover, the loss of neutrallipids was greatly reduced by the use of aqueous methanol in the firstextraction step. As seen in FIG. 5B, methanol extraction at highertemperatures improved neutral lipid purity but slightly decreased thetotal lipid recovery in the subsequent step.

In some exemplary embodiments the temperature of the extraction processis controlled in order to ensure optimal stability of algal componentspresent in the algal biomass. Algal proteins, carotenoids, andchlorophyll are examples of algal components that exhibit temperaturesensitivity. In other embodiments, the temperature is increased afterthe temperature sensitive algal components have been extracted from thealgal biomass.

In still other exemplary embodiments, the temperature of the extractionprocess is adjusted in order to optimize the yield of the desiredproduct. Extractions can be run from ambient temperature up to, butbelow, the boiling point of the extraction mixture. In still otherembodiments, the temperature of the extraction process is changeddepending on the solubility of the desired product. In still otherembodiments, the extraction temperature is optimized depending on thealgal strain of the biomass to be extracted. Elevated extractiontemperatures increase the solubility of desired compounds and reduce theviscosity of the extraction mixture enhancing extraction recovery.

In some embodiments, the extraction is run under pressure to elevate theboiling point of the extraction mixture. In these implementations, thepressure is increased to the degree necessary to prevent boiling, whilemaintaining the temperature of the extraction mixture below atemperature at which any of the desired products would begin to degrade,denature, decompose, or be destroyed.

In some exemplary embodiments, the extraction is performed near theboiling point of the solvent used, at the conditions under which theextraction is performed (e.g., atmospheric or elevated pressures). Inother embodiments, the extraction is performed near the boiling point ofthe extraction mixture, again accounting for other extractionconditions. At such temperatures, vapor phase penetration of the solventinto the algal cells is faster due to lower mass transfer resistance. Ifthe extraction temperature is allowed to significantly exceed theboiling point of the solvent, the solvent-water system can form anazeotrope. Thus, maintaining the system at or near the boiling point ofsolvent would generate enough vapors to enhance the extraction, whilereducing expense. In addition, the solubility of oil is increased athigher temperatures, which can further increase the effectiveness ofextraction at temperatures close to the solvent boiling point. FIG. 6shows the total lipid recovery in the aqueous methanol-petroleum etherextraction scheme. Although performing the methanol extraction near itsboiling temperature slightly decreases the neutral lipid recovery asobserved in FIG. 5B, it enhances the total lipid recovery.

In other embodiments, the extraction is carried out under ambientlighting conditions. In other embodiments, the extraction is carried outin an opaque container such as, but not limited to, a steel tube orcasing, in order to protect light sensitive algal components fromdegradation. Carotenoids are light sensitive algal components.

In other exemplary embodiments, the extraction takes place under normalatmospheric conditions. In still other embodiments, the extraction takesplace under a nitrogen atmosphere in order to protect algal componentsprone to oxidation. In still other embodiments, the extraction takesplace under an atmosphere of inert gas in order to protect algalcomponents prone to oxidation. Algal components that might be prone tooxidation include carotenoids, chlorophyll, and lipids.

In exemplary embodiments, the solvent-to-solid ratio for the extractionis between 3-5 based on the dry weight of the solids in the biomass. Theresidual algal biomass is rich in carbohydrates (e.g., starch) and canbe used as a feed stock to produce the solvent used for extraction.

FIG. 7 shows the effect of the solvent to solid ratio on the total lipidrecovery. As the solvent to solid ratio was increased, there was acorresponding and drastic increase in total lipid recovery. It isbelieved that this was because of the lower solubility of lipids inmethanol as compared to other commonly used oil extraction solvents suchas hexane.

The solubility of components is affected by the polarity of solvent usedin an extraction process. The solubility properties of a desired productcan be used to determine the appropriate ratio of wet biomass to solventin order to selectively extract the desired product. In the instant twosolvent extraction system, the hydrophobic solvent amount is calculatedbased on the solubility of the amphipathic solvent and lipids in thehydrophobic solvent.

A polarity index of between about 6.5 to 6.7 extracts proteins and otheralcohol soluble materials such sugars and salts. It has been found thatafter the removal of alcohol and proteins from such an extractionmixtures, the remaining material is an excellent fermentation medium.The polarity index of the mixture for extraction of polar lipids andneutral lipids is calculated to be between about 5.6 to 5.9 and 5.3 to5.5 respectively. The key to extracting the desired components is tomanipulate the polarity of the extraction mixture by varying the amountsof hydrophobic and amphipathic solvents, always accounting for the waterpresent in the wet algal biomass. By using the known polarity index of asolvent, the amount of the solvents can be adjusted to achieve thedesired extraction mixture polarity.

For example, a 40% w/w wet biomass has 40 g biomass and 60 g water forevery 100 g of wet biomass. If 100 g of ethanol is added to thismixture, the ratio of ethanol to wet biomass is 1 part wet biomass to 1part ethanol and the concentration of ethanol in the mixture is100/(100+60) equals about 62% w/w of ethanol in the liquid phase. 62%w/w of ethanol in ethanol water mixture corresponds to a polarity indexof 6.6, calculated by weight and averaging the polarities of thecomponents. Ethanol, having a polarity index of 5.2, and water, having apolarity index of 9, in a mixture containing 62% ethanol and 38% waterresults in a polarity index of (0.62*5.2+0.38*9) about 6.6. The polarityindex of the mixture for extraction of polar lipids and neutral lipidsis calculated to be about 5.8 and 5.4 respectively.

For example, a 40% w/w wet biomass has 40 g biomass and 60 g water forevery 100 g of wet biomass. If 100 g of ethanol is added to thismixture, the ratio of ethanol to wet biomass is 1 part wet biomass to 1part ethanol and the concentration of ethanol in the mixture is100/(100+60) equals about 62% w/w of ethanol in the liquid phase. 62%w/w of ethanol in ethanol water mixture corresponds to a polarity indexof 6.6, calculated by weight and averaging the polarities of thecomponents. Ethanol, having a polarity index of 5.2, and water, having apolarity index of 9, in a mixture containing 62% ethanol and 38% waterresults in a polarity index of (0.62*5.2+0.38*9) about 6.6. The polarityindex of the mixture for extraction of polar lipids and neutral lipidsis calculated to be about 5.8 and 5.4 respectively. If acetonitrile isused as the hydrophobic solvent, the amount of acetonitrile would bebetween about 2% and 4% of the amount of ethanol, because there would bean interfacial layer volume almost twice the amount of water in theethanol/acetonitrile/water azeotrope, which is about 2%.

In another example, a 40% w/w wet biomass has 40 g biomass and 60 gwater for every 100 g of wet biomass. If 100 g of ethanol is added tothis mixture, the ratio of ethanol to wet biomass is 1 part wet biomassto 1 part ethanol and the concentration of ethanol in the mixture is100/(100+60) equals about 62% w/w of ethanol in the liquid phase. 62%w/w of ethanol in ethanol water mixture corresponds to a polarity indexof 6.6, calculated by weight and averaging the polarities of thecomponents. Ethanol, having a polarity index of 5.2, and water, having apolarity index of 9, in a mixture containing 62% ethanol and 38% waterresults in a polarity index of (0.62*5.2+0.38*9) about 6.6. The polarityindex of the mixture for extraction of polar lipids and neutral lipidsis calculated to be about 5.8 and 5.4 respectively. If hexane is used asthe hydrophobic solvent, the amount of hexane would be between about 6%and 12% of the amount of ethanol, because there would be an interfaciallayer volume almost twice the amount of water in theethanol/hexane/water azeotrope, which is about 6%. This is because thesize of the interfacial layer is related to the miscibility of the twosolvents used in the system, and is further affected by thedetergent-like effects of the proteins and various lipids present in theextraction mixture. The size of the interfacial layer is also related tothe temperature of the mixture. As the temperature is increased, the twosolvents become more miscible, thereby increasing the size of theinterfacial layer.

In another example, if the extraction solvent is a 1:1 mixture ofisopropyl alcohol and ethanol, the polarity of this solvent is((3.9+5.4)/2) which is about 4.65. The ratio of solvent to wet biomasswould be calculated to match the polarities. To get a 6.6 polarityindex, we would need to make a 55% w/w of IPA-water mixture calculatedby solving the following algebraic equation:

$\begin{matrix}{{{x*4.65} + {\left( {1 - x} \right)*9}} = 6.6} \\ \downarrow \\{x = {{\left( {9 - 6.6} \right)/\left( {9 - 4.65} \right)} = 0.55}} \\ \downarrow \\{55\%\mspace{14mu} w\text{/}w\mspace{14mu}{of}\mspace{14mu}{solvent}\mspace{14mu}{mix}\mspace{14mu}{in}\mspace{14mu}{solvent}\mspace{14mu}{mix}\text{-}{water}}\end{matrix}$

Therefore, for a 40% w/w wet biomass, the wet biomass to IPA ratio is(1˜0.55)/0.6˜0.75.

With a 40% w/w wet biomass this would correspond to a ratio of 100 partswet biomass to 75 parts solvent mixture. A 40% w/w wet biomass has 40 gbiomass and 60 g water for every 100 g of wet biomass. If 75 g ofsolvent mixture is added to this mixture, the concentration of solventin the mixture is (75/(75+60)) is about 55% w/w of solvent mixture inthe solvent mixture-water solution. These calculations can be used toobtain the solvent biomass ratio at each extraction stage and for eachproduct. A few nonlimiting examples of solvent sets appear in Table 3Aand 3B.

TABLE 3A Extraction Solvent- solvent parts wet parts Biomass Parts DryParts water polarity biomass solvent dryness Biomass Water ratio indexOne step Protein Extraction Ethanol 100 99 40% 40 60 0.62 5.2 IPA 100 5240% 40 60 0.46 3.9 MeOH 100 93 40% 40 60 0.61 5.1 Propanol 100 54 40% 4060 0.47 4 1:1 IPA EtOH mixture 100 68 40% 40 60 0.53 4.55 95% ethanolwater mixture 100 115 40% 40 60 0.66 5.39 95% ethanol 5% methanolmixture 100 99 40% 40 60 0.62 5.195 95% ethanol 5% IPA mixture 100 9540% 40 60 0.61 5.135 One step Polar lipids Extraction Ethanol 100 32040% 40 60 0.84 5.2 IPA 100 101 40% 40 60 0.63 3.9 MeOH 100 274 40% 40 600.82 5.1 Propanol 100 107 40% 40 60 0.64 4 1:1 IPA EtOH mixture 100 15440% 40 60 0.72 4.55 95% ethanol water mixture 100 468 40% 40 60 0.895.39 95% ethanol 5% methanol mixture 100 317 40% 40 60 0.84 5.195 95%ethanol 5% IPA mixture 100 289 40% 40 60 0.83 5.135 One step Neutrallipids Extraction Ethanol 100 1,080 40% 40 60 0.95 5.2 IPA 100 144 40%40 60 0.71 3.9 MeOH 100 720 40% 40 60 0.92 5.1 Propanol 100 154 40% 4060 0.72 4 1:1 IPA EtOH mixture 100 254 40% 40 60 0.81 4.55 95% ethanolwater mixture 100 21,600 40% 40 60 1.00 5.39 95% ethanol 5% methanolmixture 100 1,054 40% 40 60 0.95 5.195 95% ethanol 5% IPA mixture 100815 40% 40 60 0.93 5.135

TABLE 3B Extraction Solvent- solvent Solution parts wet parts BiomassParts Dry Parts water polarity polarity biomass solvent dryness BiomassWater ratio index index One step Protein Extraction Ethanol 100 99 40%40 60 0.62 5.2 6.63 IPA 100 52 40% 40 60 0.46 3.9 6.63 MeOH 100 93 40%40 60 0.61 5.1 6.63 Propanol 100 54 40% 40 60 0.47 4 6.63 1:1 IPA EtOHmixture 100 68 40% 40 60 0.53 4.55 6.63 95% ethanol water mixture 100115 40% 40 60 0.66 5.39 6.63 95% ethanol 5% methanol mixture 100 99 40%40 60 0.62 5.195 6.63 95% ethanol 5% IPA mixture 100 95 40% 40 60 0.615.135 6.63 One step Polar lipids Extraction Ethanol 100 320 40% 40 600.84 5.2 5.80 IPA 100 101 40% 40 60 0.63 3.9 5.80 MeOH 100 274 40% 40 600.82 5.1 5.80 Propanol 100 107 40% 40 60 0.64 4 5.80 1:1 IPA EtOHmixture 100 154 40% 40 60 0.72 4.55 5.80 95% ethanol water mixture 100468 40% 40 60 0.89 5.39 5.80 95% ethanol 5% methanol mixture 100 317 40%40 60 0.84 5.195 5.80 95% ethanol 5% IPA mixture 100 289 40% 40 60 0.835.135 5.80 One step Neutral lipids Extraction Ethanol 100 1,080 40% 4060 0.95 5.2 5.40 IPA 100 144 40% 40 60 0.71 3.9 5.40 MeOH 100 720 40% 4060 0.92 5.1 5.40 Propanol 100 154 40% 40 60 0.72 4 5.40 1:1 IPA EtOHmixture 100 254 40% 40 60 0.81 4.55 5.40 95% ethanol water mixture 10021,600 40% 40 60 1.00 5.39 5.40 95% ethanol 5% methanol mixture 1001,054 40% 40 60 0.95 5.195 5.40 95% ethanol 5% IPA mixture 100 815 40%40 60 0.93 5.135 5.40

The extraction mixture described in all examples, is made up of asubstantially solid phase and a substantially liquid phase. These phasesare then separated post extraction. This can then be followed by removalof the liquid solvent from the liquid phase, yielding an extractionproduct. In some embodiments, the solvent is evaporated. In such animplementation, a liquid-liquid extraction technique can be used toreduce the amount of solvent that needs to be evaporated. Any solventsused can be recycled if conditions allow.

It was theorized that treatment of the algal biomass prior to extractionwould enhance the productivity and efficiency of lipid extraction. Inthis direction an experiment was done comparing the effect of adding abase or another organic solvent to an algal biomass to change thesurface properties and enhance extraction. A variety of treatmentsincluding aqueous methanol, aqueous sodium hydroxide, and aqueous DMSOwere attempted. As FIG. 8 demonstrates, the addition of 5% DMSOincreases the lipid recovery 3-fold. These extraction steps may beexploited to dramatically reduce the methanol extraction steps. However,the solutions used in the above experiments may not be ideal for use onlarger scales due to the high cost, viscosity, and ability to recoverand recycle DMSO.

FIG. 9 is a chart showing the effect of an eight step methanolextraction on the cumulative total lipid yield and the purity of theextracted neutral lipid. In this embodiment, 112 grams of wet biomass(25.6% dry weight), was extracted with 350 mL pure methanol and heatingfor 10 minutes at 160 W irradiance power in each step. This resulted inan extraction temperature of about 75° C., which was near the boilingpoint of the extraction mixture. Using this process, it was determinedthat it is possible to obtain highly pure neutral lipids from algal oilonce the majority of the polar lipids have been extracted. FIG. 9 showsthat it is possible to isolate high purity neutral lipid once the polarlipids are all extracted. In this case a 5% yield of total biomass wasachieved with over 90% neutral lipids purity in methanol extractionsteps 5 through 8. Furthermore, due to the boiling point of theextraction mixture, most of the water in the biomass is completelyextracted in the first extraction step, along with carbohydrates,proteins and metals.

FIG. 10 shows that recovery of lipids can be made more efficient by theuse of ethanol to extract lipids and protein from wet biomass. By usingethanol, 80% total lipid recovery can be achieved in about 4 stepsrather than the 9 generally needed by using methanol. This increase inrecovery may be attributed to greater solubility of lipids in ethanol ascompared to methanol. Furthermore, the boiling point of aqueous ethanolis higher than aqueous methanol, facilitating further recovery oflipids. This is because the higher temperature renders the oil lessviscous, thereby improving diffusability. Another distinct advantage ofthis process is using the residual ethanol in the oil fraction fortransesterification as well as lowering the heat load on the biomassdrying operation.

Further, FIG. 10 demonstrates that the initial fractions are non-lipidrich, containing proteins and other highly polar molecules, followed bythe polar lipid rich fractions and finally the neutral lipid fractions.Hence with a proper design of the extraction apparatus, one can recoverall the three products in a single extraction and fractionation process.

Another embodiment of the current invention utilizes microwaves toassist extraction. Based on previously gathered data disclosed in thisapplication, it is shown that methanol is the best single solvent forextraction of all lipids from algae. Hence, a single solvent multiplestep extraction, as described in Example 1 of the instant application,was performed in order to gather data on the efficacy of a one solventmicrowave extraction system.

FIG. 11 is a logarithmic plot comparing the extraction time and totallipid recovery of conventional extraction and microwave-assistedextraction. Based on the slope of the curve, it was calculated that themicrowave system reduces the extraction time by about five fold or more.While the conventional methods have a higher net lipid recovery, this isdue to higher recoveries of polar lipids. Based on these results, theconditions for extraction of dry algal biomass using solvents with andwithout microwave assistance have been optimized. Some embodiments ofthe invention use traditional microwave apparatus, which emitwavelengths that excite water molecules. Further embodiments of theinvention utilize customized microwave apparatus capable of excitingdifferent solvents. Still other embodiments of the invention utilizecustom microwave apparatus capable of exciting the lipids present in thealgal biomass. In some embodiments, the lipids present in the algalbiomass are excited using microwaves, thereby enhancing the separationand extraction of the lipid components from the algal biomass.

Moisture content is another parameter of biomass that will influence theefficiency of oil extraction. In some embodiments of the presentinvention, dry algal mass is extracted and fractionated. In otherembodiments, the algal mass is wet. Biomass samples with algae masscontents of 10%, 25%, and 33% were used to investigate the influence ofmoisture on extraction performance.

FIG. 12A shows an illustrative process 400 for a step-wise extraction ofproducts from an algae biomass. All units in FIG. 12A are in pounds.FIG. 12A shows a mass balance of the process 400, while the details ofthe equipment and/or systems for performing the process are describedelsewhere herein. A biomass containing 5 pounds of algae has about 0.63pounds of polar lipids, 1.87 pounds neutral lipids, 1 pound protein, and1.5 pounds carbohydrates. The biomass and 1000 pounds of water isprocessed in a dewatering step 405, which separates 950 pounds of waterfrom the mixture and passes 5 pounds of algae in 45 pounds of water to afirst extraction step 410. Any of the dewatering techniques disclosedherein can be used tin dewatering step 405. In the first extraction step410, 238 pounds of ethanol and 12 pounds of water are combined with thealgae and water from the previous step. The first extraction step 410has a liquid phase of about 80.9% w/w ethanol. A first liquid phase of231 pounds of ethanol, 53 pounds of water, and 0.5 pounds of algalproteins are recovered, from which water and ethanol are removed by,e.g., evaporation, leaving a protein-rich product 415. Solvent recoveredfrom the evaporation can be recycled to the first extraction step 410.

A first solid phase from the first extraction step 410 is passed to asecond extraction step 420; this first solid phase includes 4.5 poundsof algae, 2.6 pounds of water, and 10.9 pounds of ethanol. Eighty-sixpounds of ethanol and 4 pounds of water are added to the first solidphase from the previous step. The second extraction step 420 has aliquid phase of about 93.6% w/w ethanol. A second liquid phase of 85.9pounds ethanol, 5.9 pounds water, and 0.6 pounds polar lipids arerecovered, from which water and ethanol are removed by, e.g.,evaporation, leaving a polar lipid-rich product 425. Solvent recoveredfrom the evaporation can be recycled to the second extraction step 420.

A second solid phase from the second extraction step 420 is passed to athird extraction step 430; this first solid phase includes 3.9 pounds ofalgae, 0.7 pounds of water, and 11 pounds of ethanol. Seventy-found anda half pounds of ethanol and 3.5 pounds of water are added to the secondsolid phase from the previous step. The third extraction step 430 has aliquid phase of about 95.4% w/w ethanol. A third liquid phase of 78.9pounds ethanol, 3.9 pounds water, and 1.6 pounds neutral lipids arerecovered, from which water and ethanol are removed by, e.g.,evaporation, leaving a neutral lipid-rich product 435. Solvent recoveredfrom the evaporation can be recycled to the second extraction step 430 Asolid phase of 2.3 pounds algae, 0.3 pounds water, and 6.6 poundsethanol remain.

As demonstrated in FIG. 12A, the resulting lipid profile with eachsequential ethanol extraction step was largely influenced by themoisture content in the starting algae. Models of process 400 were runon three different biomass collections, each having a different initialwater content. As the initial water content decreased, the maximum lipidrecovery step changed from the third extraction step to a fourth (notshown). However, the overall lipid recovery from these three biomasssamples were quite similar, all above 95% of the total lipid content ofthe algal biomass.

When algal mass with higher moisture content was used, the ethanolconcentration in the aqueous ethanol mixture was much lower, andconsequently the neutral lipid percentage in the crude extract was alsolower. It has been reported that dewatering an algae paste with 90%water is a very energy intensive process. The methods described hereinunexpectedly can be used to successfully extract and fractionate analgal mass containing mostly water. As overall lipid recovery was notsignificantly influenced by starting from an algae paste containing 90%water (10% algal solids), unlike conventional extraction methods, themethods disclosed herein do not require the use of an energy intensivedrying step.

FIG. 12B shows an illustrative implementation 500 of one of theextraction steps of process 400. An algae biomass and solvent mixture505 is provided to an extraction vessel 510. After the algae isextracted (as described elsewhere herein), the mixture is provided to acoarse filtration system 515, such as a sintered metal tube filter,which separates the mixture into a liquid phase and a solid phase. Thesolid phase is passed to a downstream extraction step. The liquid phaseis passed to a solvent removal system 520, e.g., an evaporator, toreduce the solvent (e.g., ethanol) content in the liquid phase. Theliquid phase remaining after solvent removal is, optionally, passed to acentrifuge 525. Any solids remaining in the solvent removal system arerecycled or discarded. Centrifuge 525 assists in separating the desiredalgal product (e.g., proteins or lipids) from any remaining water and/orsolids in the liquid phase.

FIG. 14 shows an example of a process 600 by which an algal mass can beprocessed to form or recover one or more algal products. In thisexample, an algal biomass is extracted in a step-wise manner in afront-end process 605 using the methods disclosed herein. The extractionand separation steps are followed by an esterification process 610, ahydrolysis process 615, a hydrotreating process 620, and/or adistillation process 625 to further isolate components and products. Thecomponents and products include algal lipids, algal proteins, glycerine,carotenoids, nutraceuticals (e.g., long chain unsaturated oils and/oresters), fuel esters (generally, the esters having chain lengths of C20or shorter), fuels, fuel additives, naphtha, and/or liquid petroleumsubstitutes. In preferred embodiments the fuel esters are C16 chainlengths. In others, the fuel esters are C18 chain lengths. In stillother embodiments, fuel esters are a mixture of chain lengths, C20 orshorter.

The esterification process 610, hydrolysis process 615, hydrotreatingprocess 620, and distillation process 625 are optional and can be usedin various orders. The dashed arrows and dotted arrows indicate some,but not all, of the options for when the hydrolysis, hydrotreating,and/or distillation processes may be performed in the processing of thelipid fractions. For example, in some embodiments of the invention,after extraction and/or separation are carried out, the neutral lipidsfraction can be directly hydrotreated in order to make fuel productsand/or additives. Alternatively, in other embodiments, the neutral lipidfraction can be passed to esterification process 610.

Esterification process 610 can include techniques known in the art, suchas acid/base catalysis, and can include transesterification. Althoughbase catalysis is not excluded for producing some products, acidcatalysis is preferred as those techniques avoid the soaps that areformed during base catalysis, which can complicated downstreamprocessing. Enzymatic esterification techniques can also be used.Esterification can process substantially pure lipid material (over 75%lipid, as used herein). After esterification, glycerine byproduct can beremoved. The esterified lipids can then undergo molecular and/ornonmolecular distillation (process 625) in order to separate esterifiedlipids of different chain lengths as well as carotenoids present in thelipid fraction. The esterified lipids can then be passed tohydrotreating process 620 to generate jet fuel, biodiesel, and otherfuel products. Any hydrotreating process known in the art can be used;such a process adds hydrogen to the lipid molecules and removes oxygenmolecules. Exemplary conditions for hydrotreating comprise reacting thetriglycerides, fatty acids, fatty acid esters with hydrogen under highpressure in the range of 600 psi and temperature in the range of 600° F.Commonly used catalysts are NiMo or CoMo.

Hydrotreating the fuel esters rather than the raw lipids has severaladvantages. First, the esterification process 610 reduces the levels ofcertain phosphorus and metals compounds present in algal oils. Thesematerials are poisons to catalysts typically used in hydrotreatingprocesses. Thus, esterification prior to hydrotreating prolongs the lifeof the hydrotreating catalyst. Also, esterification reduces themolecular weight of the compounds being hydrotreated, thereby improvingthe performance of the hydrotreating process 620. Further still, it isadvantageous to retain the fuel esters from the distillation process 625to be hydrotreated in a vaporous form, as doing so reduces the energyneeded for hydrotreating.

In some embodiments of the invention, the neutral algal lipids aredirectly hydrotreated in order to convert the lipids into fuel productsand additives. While in other implementations, the neutral lipids areesterified and separated into carotenoids, long chain unsaturatedesters, eicosapentaenoic acid (EPA) esters, and/or fuel esters viadistillation process 625. Distillation process 625 can include moleculardistillation as well as any of the distillation techniques known in theart. For example, the distillates can be fractionated using a simpledistillation column to separate the lower chain fatty acids forrefining. The long chain unsaturated fatty acids remain as high boilingresidue in the column. In some embodiments, the remaining vapor can thenbe sent to the hydrotreating process. Two of the advantages of thepresent invention are that it yields pure feed as well as a vaporproduct, which favors the energy intensive hydrotreating reaction, asdescribed above.

In some embodiments of the invention, polar lipids (and, optionally,neutral lipids) are hydrolyzed in hydrolysis process 615 before beingpassed to the esterification process. Doing so unbinds the fatty acidsof the algal lipids, and enables a greater amount of the algal lipids tobe formed into useful products.

FIG. 15 is a flowchart showing a process 700 for producing nutraceuticalproducts from neutral lipids. In one implementation of process 700,neutral lipids are fed to an adsorption process 705 that separatescarotenoids from EPA-rich oil. The neutral lipids can be from an algaesource generated by any of the selective extraction techniques disclosedherein. However, the neutral lipids can be from other sources, such asplant sources.

Adsorption process 705 includes contacting the neutral lipids with anadsorbent to adsorb the carotenoids, such as beta carotene andxanthophylls. In one implementation, the adsorbent is Diaion HP20SS(commercially available from ITOCHU Chemicals America, Inc.). Theneutral lipids can contact the adsorbent in a batch-type process, inwhich the neutral lipid and adsorbent are held in a vessel for aselected amount of time. After the contact time, the absorbent andliquid are separated using techniques known in the art. In otherimplementations, the adsorbent is held in an adsorbent bed, and theneutral lipids are passed through the adsorbent bed. Upon passingthrough the adsorbent bed, the carotenoids content of the neutral lipidsis reduced, thereby producing an oil rich in EPA.

The carotenoids can be recovered from the adsorbent material by treatingthe adsorbent with an appropriate solvent, including, but not limitedto, alcohols such as ethanol, isopropyl alcohol, butanol, esters such asethyl acetate or butyl acetate, alkanes such as hexane, and pentane.

FIG. 16 is a flowchart showing a process 800 for producing fuel products830 from neutral lipids 805. The neutral lipids can be from an algaesource generated by any of the selective extraction techniques disclosedherein. However, the neutral lipids can be from other sources, such asplant sources. The neutral lipids are treated in a degumming process810, in which the lipids are acid washed to reduce the levels of metalsand phospholipids in the neutral lipids. In some implementations, arelatively dilute solution of phosphoric acid is added to the neutrallipids, and the mixture is heated and agitated. The precipitatedphospholipids and metals are then separated from the remaining oil, forexample, by centrifuge.

The treated oil is then passed to bleaching process 815 to removechlorophylls and other color compounds. In some implementations,bleaching process 815 includes contacting the oil with clay and or otheradsorbent material such as bleaching clay (i.e. bentonite or fuller'searth), which reduce the levels of chlorophylls and other colorcompounds in the oil. The treated oil then is passed to hydrotreatingprocess 820, which hydrogenates and deoxygenates the components of theoil to form fuels products, for example, jet fuel mixtures, diesel fueladditive, and propane. In addition, the hydrotreating process 820 alsocauses some cracking and the creation of smaller chain compounds, suchas LPG and naptha. Any of the hydrotreating processes described hereincan be used for hydrotreating process 820.

The mixture of compounds created in the hydrotreating process 820 arepassed to a distillation process 825 to separate them into various fuelproducts 830. Distillation process 825 can include any of the molecularand non-molecular distillation techniques described herein or known inthe art for separation of fuel compounds.

In some embodiments of the instant invention, proteins may beselectively extracted from an algal biomass. Extraction of proteinsusing the disclosed methods offers many advantages. In particular, algalcells do not need to be lysed prior to extracting the desired proteins.This simplifies and reduces costs of extraction. The methods of theinstant invention exploit the solubility profiles of different classesof proteins in order to selectively extract and fractionate them from analgal culture, biomass, paste, or cake.

For example, an algal biomass may be subjected to heating and mixing toextract water and salt soluble proteins called albumins and globulins.This mixture can then be subjected to a change in pH to recover thealkali soluble proteins called the glutelins. This step can then befollowed by a solvent-based separation of the alcohol soluble proteinscalled prolamins. The remaining biomass would be rich in carbohydratesand lipids.

Proteins can be extracted from both saltwater and freshwater algalcells, as shown in FIGS. 17 and 18. The presence of salt in thesaltwater algal culture or biomass affects the extraction of differentclasses of protein, but the methods disclosed herein enable one toselectively extract proteins from either fresh or saltwater algae.

In some embodiments, extraction of proteins from freshwater algal cellsis accomplished by the novel process shown in FIG. 17. Freshwater algalcells or a freshwater algal biomass are heated and mixed. Mixing can beaccomplished by a variety of methods known in the art such as, but notlimited to, stirring, agitation, and rocking. This process generates afirst heated extraction mixture or slurry, comprised of a firstsubstantially liquid phase and a first substantially solid phase. Thesolid and liquid phases are then separated. Separation can beaccomplished by a variety of methods known in the art including, but notlimited to, centrifugation, decantation, flotation, sedimentation, andfiltration. This first substantially liquid phase is enriched in albuminproteins.

The first substantially solid phase is then mixed with salt water andheated to generate a second heated extraction mixture or slurry,comprised of a second substantially liquid phase and a secondsubstantially solid phase. The salt water may be natural seawater or maybe an aqueous salt solution. An example of such a solution wouldcomprise about typically 35 g/L comprising mainly of NaCl. The solid andliquid phases are then separated. This second substantially liquid phaseis enriched in globulin proteins.

The second substantially solid phase is then mixed with water and heatedto generate a third heated extraction mixture or slurry, comprised of athird substantially liquid phase and a third substantially solid phase.The pH of this third extraction mixture or slurry is then raised toabout 9 or greater, enriching the third substantially liquid phase withglutelin proteins. The solid and liquid phases are then separated, thethird substantially liquid phase being enriched in glutelin proteins.

The third substantially solid phase is then mixed with a solvent set andheated to generate a fourth heated extraction mixture or slurry,comprised of a fourth substantially liquid phase and a fourthsubstantially solid phase. In one preferred embodiment, the solvent setcomprises ethanol. In other non-limiting embodiments, the solvent setcomprises one or more of the following solvents: methanol, isopropanol,acetone, ethyl acetate, and acetonitrile. The solid and liquid phasesare then separated. This fourth substantially liquid phase is enrichedin prolamin proteins. The remaining fourth substantially solid phase maybe enriched in lipids, depending on the composition of the startingalgal biomass.

In some embodiments, extraction of proteins from saltwater algal cellsis accomplished by the novel process shown in FIG. 18. Saltwater algalcells or a saltwater algal biomass are heated and mixed. Mixing can beaccomplished by a variety of methods known in the art such as, but notlimited to, stirring, agitation, and rocking This process generates afirst heated extraction mixture or slurry, comprised of a firstsubstantially liquid phase and a first substantially solid phase. Thesolid and liquid phases are then separated. Separation can beaccomplished by a variety of methods known in the art including, but notlimited to, centrifugation, decantation, flotation, sedimentation, andfiltration. This first substantially liquid phase is enriched inglobulin proteins.

The first substantially solid phase is then mixed with water and heatedto generate a second heated extraction mixture or slurry, comprised of asecond substantially liquid phase and a second substantially solidphase. The solid and liquid phases are then separated. This secondsubstantially liquid phase is enriched in albumin proteins.

The second substantially solid phase is then mixed with water and heatedto generate a third heated extraction mixture or slurry, comprised of athird substantially liquid phase and a third substantially solid phase.The pH of this third extraction mixture or slurry is then raised to pH 9or greater, enriching the third substantially liquid phase with glutelinproteins. The solid and liquid phases are then separated, the thirdsubstantially liquid phase being enriched in glutelin proteins.

The third substantially solid phase is then mixed with a solvent set andheated to generate a fourth heated extraction mixture or slurry,comprised of a fourth substantially liquid phase and a fourthsubstantially solid phase. In one preferred embodiment, the solvent setcomprises ethanol. In other non-limiting embodiments, the solvent setcomprises one or more of the following solvents: methanol, isopropanol,acetone, ethyl acetate, and acetonitrile. The solid and liquid phasesare then separated. This fourth substantially liquid phase is enrichedin prolamin proteins. The remaining fourth substantially solid phase maybe enriched in lipids, depending on the composition of the startingalgal biomass.

The disclosed methods also provide for the selective extraction ofdifferent types of proteins, as shown in FIGS. 17-20. Any of the stepsof the aforementioned extraction process can be performed separatelyfrom the rest of the steps in order to selectively extract a singleprotein product. Two examples of this appear in FIGS. 17 and 18, as theas demonstrated by the dashed box around extraction step 1 a.

In a non-limiting example, globulin proteins can be selectivelyextracted from a freshwater algal biomass by mixing said biomass withsalt water and heating to generate a heated extraction mixture orslurry, comprised of a substantially liquid phase and a substantiallysolid phase. The solid and liquid phases can then be separated. Theliquid phase is enriched in globulin proteins. See FIG. 17, extractionstep 1 a.

In another non-limiting example, albumin proteins can be selectivelyextracted from a saltwater algal biomass by mixing said biomass withwater and heating to generate a heated extraction mixture or slurry,comprised of a substantially liquid phase and a substantially solidphase. The solid and liquid phases can then be separated. The liquidphase is enriched in globulin proteins. See FIG. 18, extraction step 1a.

In a further non-limiting example, prolamin proteins can be selectivelyextracted from either a freshwater or saltwater algal biomass as shownin FIG. 19. The selective extraction is accomplished by mixing the algalbiomass with a solvent set and heating to generate a heated extractionmixture or slurry, comprised of a substantially liquid phase and asubstantially solid phase. The solid and liquid phases can then beseparated. The liquid phase is enriched in prolamin proteins.

In yet another non-limiting example, a protein fraction can beselectively extracted from either a freshwater or saltwater algalbiomass as shown in FIG. 20. The selective extraction is accomplished bymixing the algal biomass with a solvent set to generate an extractionmixture or slurry and effecting a pH change in the mixture. The mixtureis comprised of a substantially liquid phase and a substantially solidphase. The solid and liquid phases can then be separated. The liquidphase is enriched in proteins.

FIG. 22 is a schematic of an exemplary extraction system for theextraction of proteins by a single solvent (amphipathic) system. Theexemplary system comprises an algae culture (2201), which may be pumpeddirectly in or may be stored in a hold tank, a centrifuge (2202), anextractor (2203), a solids separation filter (2204), a heater (2205), asolvent recover system (2206), and a chiller (2207). Pumps (2213) assistin moving the product of each step to the next step.

FIG. 23 shows an exemplary system for extracting proteins and polarlipids from wet algal biomass, according to one exemplary embodiment.The exemplary system comprises an algae culture, which may be pumpeddirectly in or may be stored in a hold tank (2301), a centrifuge (2302),an extractor (2303), a decanter (2304), a heater (2305), a solventrecovery system (2306), a chiller (2307), a hold tank (2308), a solidseparator (2309), a second heater (2310), a second solvent recoverysystem (2311) and a second chiller (2312). Pumps (2313) assist in movingthe product of each step to the next step.

The algae culture, which may be pumped directly in or may be stored in ahold tank, (2301) is centrifuged (2302) to obtain a concentrated pastewhich is pumped to an extractor (2303). The centrifuging unit (2302) maybe replaced by a solid separator such as a membrane system or adissolved air flotation unit or a flocculation sedimentation unit, orthe like. A mixture of amphipathic and hydrophobic solvent is added tothe extractor. The extractor mixes and heats the solution for a fixedtime and at less than the boiling point of the solvent mixture. Theheating can be achieved with various methods such as heating withmicrowaves, water, steam, hot oil, or electricity. The extraction may bedone at atmospheric pressure or under pressurized conditions to enhancethe effectiveness of the extraction.

The extract is then pumped into a decanter to separate the lighter andheavier phases. This step can also be performed using membranefiltration or by centrifuging. The lighter phase comprises thehydrophobic solvent and the polar lipids. The heavier phase consists ofan amphipathic solvent, water and algae. The hydrophobic layer is pumpedthrough a heater (2305) and into a solvent recovery system (2306). Thevapors are passed through a chiller (2307) and recovered for recycle.The residue is a concentrate comprising mostly polar lipids. The heavierphase is collected in a hold tank (2308) and pumped through a solidliquid separator (2309). The solids comprise the extracted algae biomasswhich can be further extracted or dried. The liquid portion is passedthrough a second heater (2310) and into a second solvent recovery system(2311). The vapors from this system are passed through a second chiller(2312) to recover and recycle the amphipathic solvent. The water andproteins form the residue in the solvent recovery unit.

FIG. 24 is a schematic of an exemplary extraction scheme for theextraction of neutral lipids and proteins by a two-solvent(amphipathic/hydrophobic) system. The exemplary system comprises analgae culture (2401), which may be pumped directly in or may be storedin a hold tank, a centrifuge (2402), an extractor (2403), a decanter(2404), a heater (2405), a solvent recover system (2406), a chiller(2407), a hold tank (2408), a solid liquid separator (2409), a secondheater (2410), a second solvent recover system (2411) and a secondchiller (2412). Pumps (2413) assist in moving the product of each stepto the next step.

FIG. 25 is a schematic of an exemplary system for extracting proteins,polar lipids and neutral lipids from wet algal biomass, according to oneexemplary embodiment. The exemplary system comprises an algae culture(2501), which may be pumped directly in or may be stored in a hold tank,a centrifuge (2502), an extractor (2503), a decanter (2504), a heater(2505), a solvent recover system (2506), a chiller (2507), a hold tank(2508), a solid liquid separator (2509), a second heater (2510), asecond solvent recover system (2511) and a second chiller (2512).

The algae culture (2501) is centrifuged (2502) to obtain a concentratedpaste which is pumped to an extractor (2503). The centrifuging unit(2502) may be replaced by a solid separator such as a membrane system ora dissolved air flotation unit or a flocculation sedimentation unit, orthe like. A mixture of amphipathic and hydrophobic solvent is added tothe extractor. The extractor mixes and heats the solution for a fixedtime and less than the boiling point of the solvent mixture. The heatingcan be achieved with various methods such as heated with microwaves,water, steam, or hot oil or electricity. The extraction may be done atatmospheric pressure or under pressurized conditions to enhance theeffectiveness of the extraction. The extract is then pumped into adecanter to separate the lighter and heavier phases. This step can alsobe performed using membrane filtration or by centrifuging.

The lighter phase comprises of the hydrophobic solvent and the polarlipids. The heavier phase consists of a amphipathic solvent, water andalgae. The hydrophobic layer is pumped through a heater (2505) and intoa solvent recover system (2506). The vapors are passed through a chiller(2507) and recovered for recycle. The residue is a concentratecomprising mostly of the polar lipids. The heavier phase is collected ina hold tank (2508) and pumped through a solid liquid separator (2509).The solids comprise of the extracted algae biomass which can be furtherextracted or dried. The liquid portion is passed through a second heater(2510) and into a second solvent recover system (2511). The vapors fromthis unit are passed through a second chiller (2512) to recover andrecycle the amphipathic solvent. The water and proteins form the residuein the solvent recovery unit. The extracted solids from the solid liquidseparator (2509) are further extracted again with a ratio of theamphipathic solvent to the water in the system in the second extractor(2518). A second mixture of amphipathic and hydrophobic solvent is addedto the second extractor. The second extractor mixes and heats thesolution for a fixed time and less than the boiling point of the secondsolvent mixture. The second extract is then pumped into a seconddecanter (2519) to separate the lighter and heavier phases. The lighterphase comprises of the hydrophobic solvent and the neutral lipids. Theheavier phase consists of an amphipathic solvent, water and algae. Thehydrophobic layer is pumped through a heater (2520) and into a solventrecover system (2521). The vapors are passed through a chiller (2522)and recovered for recycle. The residue is a concentrate comprisingmostly of the neutral lipids. The heavier phase is recycled to theextractor (2518) or dried. Pumps (2513) assist in moving the product ofeach step to the next step.

FIG. 26 is a schematic demonstrating the ethanol extraction concept. Asthe amount of ethanol is changed in proportion to the amount of waterand hydrophobic solvent in the system, different components can beselectively extracted.

Having been informed of these aspects of the invention, one of skill inthe art would be able to selectively extract a desired polar lipid orprotein from either a freshwater or saltwater algal biomass by either asingle step extraction process, or a multi-step extraction process. Inlight of the instant disclosure, one of skill in the art would be ableto interchange the order of the above disclosed multi-step extractionschemes, provided that the protein content of the algal mass and thesolubility properties of the proteins of interest are taken intoaccount. Other embodiments of the disclosed methods may incorporate awash step between each extraction step.

For any of the disclosed protein extraction methods, the extractionmixture/slurry may be maintained at a heated temperature for a period oftime. In some embodiments, the extraction mixture is maintained at aheated temperature for between about 20 minutes to about 90 minutes. Insome aspects, the extraction mixture is maintained at a heatedtemperature for between about 20 minutes and about 60 minutes. In otheraspects, the extraction mixture is maintained at a heated temperaturefor between about 45 minutes to about 90 minutes.

In some embodiments, the extraction mixture/slurry may be heated totemperatures less than about 50° C. In some aspects, the albumin,globulin, and glutelin proteins are extracted at temperatures of lessthan about 50° C. In other embodiments the extraction mixture/slurry isheated to a temperature close to the boiling point of extractionmixture/slurry. In some aspects, the prolamin proteins are extracted attemperatures close to the boiling point of the extractionmixture/slurry. In other embodiments, the pressure is increased aboveatmospheric pressure, up to and including, 50 psi, during the heatingand mixing steps to enhance extraction.

The shelf life of an algal biomass can be increased by removal ofinterstitial and extracellular water. This results in less microbialgrowth and contamination of the biomass over a period of time. This canbe achieved by mixing an amphipathic solvent with an algal culture orbiomass under unheated conditions. No products are extracted by thisprocess, but the water is removed from the biomass or culture andreplaced with the solvent. In some embodiments, the solvent replaces50-90% of the water in the algal culture or biomass. In some embodimentsthe amphipathic solvent is ethanol, acetone, methanol, isopropanol,butanone, dimethyl ether, and propionaldehyde. 2-propanol, acetonitrile,t-butyl alcohol, 1-propanol, water, heavy (D₂O), ethylene glycol,glycerin, or a combination thereof. In some embodiments the amphipathicsolvent is ethanol denatured with isopropanol. In other embodiments, thesolvent is isopropanol. In still other embodiments, the solvent isaqueous. In still further embodiments, the removal of interstitial andextracellular water is performed under chilled conditions. The reductionin water content in the material leads to the enhanced shelf life.

Example 1

Green microalgae Scendesmus dimorphus (SD) were cultured in outdoorpanel photobioreactors. SD samples of varying lipid contents wereharvested. After removal of bulk water by centrifugation, the algalsamples were stored as 3-5 cm algae cakes at −80° C. until use. Apre-calculated amount of wet algal biomass (15 grams dry algae weightequivalent) and 90 mL of ethanol solvent was added into a three-neckflask equipped with condenser, mechanical stirring and a thermocouple.In one experiment, the mixture was refluxed for 10 min under microwaveirradiance. In a second, the mixture was refluxed for lh with electronicheating. Afterwards, the mixture was cooled to room temperature andseparated into a diffusate and retentate by filtration.

The total lipids of algal samples were analyzed using achloroform-methanol-water system according to Bligh and Dyer's lipidextraction method. This total lipid value was used as reference for thelipid recovery calculation. Total lipids were further separated intoneutral lipids and polar lipids by standard column chromatography methodusing 60-200 mesh silica gel (Merck Corp., Germany). Each lipid fractionwas transferred into a pre-weighed vial, initially evaporated at 30° C.using a rotary evaporator (Büchi, Switzerland) and then dried under highvacuum. The dried retentates were placed under nitrogen and thenweighed. The fatty acid profile of each sample was quantified by GC-MSafter derivatization into fatty acid methyl esters using heptadecanoicacid (C17:0) as the internal standard.

The results (data not shown) indicated that microwave assistedextraction was best for removal of the polar lipids in the firstextraction step, and somewhat less effective for the separation ofneutral lipids. Electronic heating is more consistent in extractioneffectiveness. The final yield is comparable between microwave assistedextraction and electronic heating assisted extraction, but, microwaveassisted extraction is significantly faster.

Example 2 Protein Extraction from Algal Biomass

(1) Acid Leaching: Algal biomass was soaked in water at pH 4.5 for 1hour. The samples were then centrifuged at 3000 rpm for three minutes,and the supernatant removed. The remaining solids were washed 3 timeswith dilute acid (pH 4.5) and freeze dried.

(2) Alkaline extraction: Algal biomass was soaked in water at pH 11 for1 hour. following the addition of pH-adjusted water. The samples werethen centrifuged at 3000 rpm for three minutes, and the supernatantremoved. The supernatant was neutralized with acid (pH 4.5) followingthe centrifugation. The remaining solids were washed 3 times with diluteacid (pH 4.5) and freeze dried.

The results of acid leaching and alkaline extraction are shown below inTable 4.

TABLE 4 Protein Purity Protein Yield (% weight of Process (% weight)protein yield) Alkaline Extraction 16 45 Acid Leaching 70 32.5

Protein yield was calculated on a weight basis, comparing the weight ofthe freeze dried solids to the weight of the algal biomass prior tosoaking in pH-adjusted water. Protein purity was determined by theOfficial Method of the American Oil Chemists' Society (Ba-2a-38),measuring the amount of nitrogen in the freeze dried solids of eachprocess. As proteins are an important product that adds to the value ofalgal product extraction, this information allows for the use offeedstocks with varying levels of protein in the systems and methodsdisclosed herein.

Example 3 Extraction of Proteins from Saltwater Algal Biomass

The saltwater algal culture initially made up of about 1-10% w/w solidsin saltwater was heated to 50° C. and maintained at this temperature for1 hr. The resulting slurry was centrifuged to separate the liquid phasefrom the solid phase. The liquid extract was determined to be rich inglobulin proteins (about 10% of the total proteins present in theoriginal algal biomass).

The solids were then suspended in fresh water and heated to about 50° C.and maintained for about 1 hour. The resulting slurry was centrifugedagain to separate the liquid from the solid phase. The liquid phase wasdetermined to be rich in albumin proteins (about 10% of the totalproteins present in the original algal biomass).

The solids were then suspended in ethanol to achieve a 70% w/w mixture.This mixture was heated to about 75° C. and maintained at thattemperature for about 1 hour. The resulting slurry was centrifuged toseparate the liquid from the solid phase. The liquid phase wasdetermined to be rich in albumin proteins (about 30% of the totalproteins present in the original biomass).

The solids were then suspended in alkali solution (aqueous NaOH, pH 9)and heated to about 50° C. and maintained at that temperature for about1 hour. The resulting slurry was centrifuged to separate the liquid fromthe solid phase. The liquid phase was determined to be rich in glutelinproteins (about 50% of the total proteins present in the originalbiomass).

Example 4 Step Fractionation and Extraction of Algal Biomass by Ethanol

One thousand pounds of Nannochloropsis biomass (cultured from strain202.0, obtained from Arizona State University, Laboratory for AlgaeResearch and Biotechnology, ATCC Deposit Number PTA-11048), washarvested and dewatered until algae comprised about 35% w/w and thenfinally frozen.

The extraction steps were performed in a 400 gallon jacketed kettle withhinged lids. The lids were tied down with straps and sealed withsilicone. The system also contained a mixer with a 2 horsepowerexplosion proof motor with a two blade shaft. The frozen algae materialwas emptied into the tank and an equal weight of ethanol was pumped inusing a pneumatic drum pump. The material was stirred for 15 minutes andthe jacket heated with steam to obtain the desired temperature at eachextraction step. The desired temperature is near, meaning within 3° C.of the boiling point of the mixture, but not boiling. This desiredtemperature is different at each extraction step as the boiling point ofthe mixture changes as the proportion of ethanol is changed. Uponreaching the desired temperature, the system was stirred continuouslyheld at the desired temperature for 60 minutes to ensure that thecontents of the kettle were uniformly heated.

The contents of the kettle were then pumped out of the extraction vesseland into a Sharples decanter centrifuge, using a pneumatic Viking vanepump at about 1 gallon per minute. The decanter centrifuge rotor speedwas set to about 6000 rpm. The solids were collected in an enclosedplastic drum and consisted of about 50% w/w solids to liquids. Thesesolids were returned to the kettle, where the aforementioned extractionsteps were repeated. The liquid stream from the decanter was collectedinto a feed tank was and then fed to the membrane filtration system. Themembrane used was a 0.375 ft² SS membrane manufactured by GraverTechnologies. The operating conditions were 60° C.±5° C. and with anaverage pressure gradient of 40 psi. The membrane system was backwashedabout every 15 minutes with compressed air to maintain the flux. Thepermeate collected from the membrane system was free of any particulatematter. The retentate was collected and recycled to the decanter.

This extraction and fractionation is due to the change in polarity ofthe solvent through the process in each extraction. In the extractionshown in FIG. 13, the process began with about 1000 lbs. of wet algalbiomass containing about 65% pure water (35% w/w algal solids). This wasmixed with 860 lbs. of denatured ethanol (95% ethanol and 5% methanol),resulting in a mixture containing about 55% aqueous ethanol. The solidsand liquids were separated using a decanter as described above. The wetsolid portion weighed 525 lbs. and was 40% dry mass. A total of 525 lbs.of 95% the denatured ethanol was added to the solids, resulting in amixture made up of about 85% aqueous ethanol. The solids and liquidswere separated using a decanter as described above. The solid portionweighed 354.5 lbs. and was 40% dry mass. To this mass, another 700 lbs.of denatured ethanol was added, resulting in a mixture of about 95%aqueous ethanol. The solids and liquids were separated using a decanteras described above. The resulting solids were about 40% dry mass. Thisbiomass requires 60% less energy to dry, calculated based on the latentheat of water and ethanol.

In some experiments (data not shown) other types of denatured ethanolwere tried. Denatured ethanol containing 95% ethanol and 5% isopropylalcohol was used in an extraction, but was found not to be as effectiveas 95% ethanol and 5% methanol. Use of 100% ethanol is a preferredembodiment of the present invention, but is generally not available dueto cost constraints.

The permeate stream from the membrane system was evaporated using anin-house fabricated batch still. The operating conditions were about 80°C. during the vacuum distillation. All of the ethanol in the permeatewas evaporated. These extraction steps were repeated three times,resulting in four product pools, as shown in FIG. 13. This is becausewith each extraction step, the polarity changed with the addition ofwater to the mixture, allowing for the extraction of differentcomponents with each step. Product 1 contained the algal proteins, andas a result, retained excess water in the system that could not bevaporized under the operating conditions. Product 2 contained the polarlipids. Product 3 contained the neutral lipids. Finally, Product 4 wasthe residual biomass, containing potential coproducts such ascarotenoids.

Example 5 Dewatering and Extraction of Algal Biomass by Ethanol

Upon harvesting, algal biomass typically contains between about 0.1 to0.5% (w/w) solids. This can be dewatered using any of the methods knownin the algae industry, including, but not limited to membranefiltration, centrifugation, heating, sedimentation or flotation.Flocculation can either assist in flotation or sedimentation. Thetypical result of such methods is an algae slurry containing about 10%w/w solids. To dewater further, another dewatering method may be used toremove some of the remaining free water to get the concentration ofsolids closer to 40% w/w. However, the cost of dewatering increasesexponentially after the first dewatering is carried out. An advantage ofthe systems and methods disclosed herein is that the allow for theextraction and fractionation of an algal mass that has undergone onlyone round of dewatering.

An example of such a process might be that in the first extractionround, following the protocol described in Example 3, 1000 lbs. of wetbiomass containing 90% pure water and is mixed with 1000 lbs. ofdenatured ethanol (95% EtOH and 5% MeOH), resulting in a solvent mixtureof about 50% aqueous ethanol. The resulting biomass (350 lbs.) is 40%dry. The solvent composition of these wet solids is 50% aqueous ethanol.With another 350 lbs. of denatured ethanol, the composition of themixture would be about 81% aqueous ethanol. The resulting biomass (235lbs.) is 40% dry. The solvent composition of these wet solids is 81%aqueous ethanol. With another 470 lbs. of denatured ethanol, thecomposition of the mixture would be about 95% aqueous ethanol. Theresulting solids would be 40% dry with about 95% ethanol. This wetbiomass requires 60% less energy to dry based on the latent heat ofwater and ethanol. In this case, 100 lbs. of algae would have beenextracted using 1820 lbs. ethanol. When compared with Example 3, whereinthe starting material was 40% algal solids, 350 lbs. of the dry algaeequivalent was extracted with 2085 lbs. ethanol.

REFERENCES

The following references are herein incorporated by reference in theirentirety:

-   U.S. Pat. No. 7,148,366-   Rhodes, Science Progress, 92(1):39-90, 2009. Generic review on using    algae to produce biodieselChisti, Y. (2007). Biodiesel from    microalgae. Biotechnol Adv 25, 294-306. Generic review on using    algae to produce biodiesel-   Amin, Energy Conyers. Manage., 50:1834-1840, 2009. Generic review on    using algae to produce biofuel and gas-   Catchpole et al., J. of Supercritical Fluids, 47:591-597, 2009. SCF    CO2 based extraction of specialty lipids-   Bligh E G & Dyer W J. “A rapid method of total lipid extraction and    purification.” Can. J. Biochem. Physiol. 37: 911-917, 1959.-   Christie, W. W., Lipid Analysis, 3rd ed., Oily Press, Bridgewater,    UK, 2003, 416.-   Approved Methods of the AACC, 9th ed., American Association of    Cereal Chemists. St. Paul, Minn., 1995 AACC Method 58-19.-   Snyder, L. R. “Classification of the Solvent Properties of Common    Liquids” Journal of Chromatography, 92(2): 223-230 (May 1974)

1. A method of separating proteins from intact algal cells, comprising:providing a wet algal biomass comprising intact algal cells whichcomprise water, proteins that are soluble in a mixture of an amphipathicsolvent and water, and polar lipids; dewatering the wet algal biomass byremoving extracellular water to increase the solid content of the wetalgal biomass to between 5% and 50% to result in a partially dewateredalgal biomass comprising intact algal cells; mixing the partiallydewatered wet algal biomass with an amphipathic solvent set in a ratioof about one part amphipathic solvent set to about one part partiallydewatered wet algal biomass by weight, and a hydrophobic solvent set togenerate an extraction mixture comprising a heavier phase and a lighterphase, wherein the heavier phase comprises the water, amphipathicsolvent set, partially dewatered wet algal biomass that comprises intactalgal cells, and proteins that are soluble in a mixture of anamphipathic solvent and water, and the lighter phase comprises thehydrophobic solvent set and polar lipids; separating the heavier phaseof the extraction mixture from the lighter phase of the extractionmixture; separating the partially dewatered wet algal biomass from theheavier phase of the extraction mixture to generate a protein mixturecomprising proteins that are soluble in a mixture of an amphipathicsolvent and water, water, and the amphipathic solvent set; andseparating the proteins of the protein mixture from at least a portionof the water and amphipathic solvent to generate a protein fraction,wherein the protein mixture has a higher concentration of proteins thatare soluble in a mixture of an amphipathic solvent and water than theseparated partially dewatered wet algal biomass.
 2. The method of claim1 wherein dewatering is by centrifuging, filtering, settling or floatfractionating.
 3. The method of claim 1 wherein the amphipathic solventset is added in an amount to adjust the polarity index of the extractionmixture to between about 6.5 to 6.7 prior to the addition of thehydrophobic solvent set.
 4. The method of claim 3 wherein thehydrophobic solvent set is added in an amount to adjust the polarityindex of the extraction mixture to between about 5.7-5.9 after theaddition of the amphipathic solvent set.
 5. The method of claim 1wherein the amphipathic solvent set is acetone, methanol, ethanol,isopropanol, butanone, dimethyl ether, and propionaldehyde. 2-propanol,acetonitrile, t-butyl alcohol, 1-propanol, water, heavy (D₂O), ethyleneglycol, glycerin or a combination thereof.
 6. The method of claim 1wherein the hydrophobic solvent set is propane, butane, pentane, butene,propene, naphtha, an alkane, hexane, pentane, heptane, octane, an ester,ethyl acetate, butyl acetate, a ketone, methyl ethyl ketone, methylisobutyl ketone, an aromatic, toluene, benzene, cyclohexane,tetrahydrofuran, a haloalkane, chloroform, trichloroethylene, an ether,diethyl ether, diesel, jet fuel, gasoline and mixtures thereof.
 7. Themethod of claim 1, wherein the heavier phase is separated from thelighter phase by decanting, membrane filtering or centrifuging.
 8. Themethod of claim 1, wherein the wet algal biomass is separated from themixture of algal biomass with an amphipathic solvent set, protein andwater by decanting, membrane filtering or centrifuging.
 9. The method ofclaim 1 wherein the amphipathic solvent set and water are removed fromthe protein mixture by evaporation or distillation.
 10. The method ofclaim 1 further comprising recovering the amphipathic solvent set. 11.The method of claim 1 further comprising condensing and recovering theamphipathic solvent set.
 12. The method of claim 1, wherein theextraction mixture is heated.
 13. The method of claim 12, wherein themixture is heated with microwaves, water, steam, or hot oil orelectricity.
 14. The method of claim 12, wherein the extraction mixtureis heated at atmospheric pressure.
 15. The method of claim 12, whereinthe extraction mixture is heated in a pressurized reactor.
 16. Themethod of claim 15, wherein the pressurized reactor is a batch or acontinuous reactor.
 17. A method of separating proteins from intactalgal cells, comprising: providing a wet algal biomass comprising intactalgal cells which comprise water, proteins that are soluble in a mixtureof an amphipathic solvent and water, and polar lipids; mixing the wetalgal biomass with an amphipathic solvent set in a ratio of about onepart amphipathic solvent to about one part partially dewatered wet algalbiomass by weight, and a hydrophobic solvent set to generate anextraction mixture comprising a heavier phase and a lighter phase,wherein the heavier phase comprises the water, amphipathic solvent set,wet algal biomass that comprises intact algal cells, and proteins thatare soluble in a mixture of an amphipathic solvent and water, and thelighter phase comprises the hydrophobic solvent set and polar lipids;separating the heavier phase of the extraction mixture from the lighterphase of the extraction mixture; separating the wet algal biomass fromthe heavier phase of the extraction mixture to generate a proteinmixture comprising proteins that are soluble in a mixture of anamphipathic solvent and water, water, and amphipathic solvent set; andseparating the proteins of the protein mixture to generate a proteinfraction, wherein the protein mixture has a higher concentration ofproteins that are soluble in a mixture of an amphipathic solvent andwater than the separated partially dewatered wet algal biomass.